RAPD Patterns Research Articles

Molecular Method for the Detection of Resistance Aspergillus Fumigatus to Some Antifungal Agents

Twenty samples were obtained from Aspergillus fumigatus, out of a total of 45 phlegm samples 41 samples were positive for A. fumigatus patients aged (33 to 82 years of both sexes), from August 2022 to December 2022 from the Specialist Center Samples were collected from several Iraqi governorates from the north, center and south. The results indicated that the isolation of AFU1, AFU12 and AFU31 was the highest resistance to the antihistamines. Anti-fungal resistance, most of which have allergic reactions to these antibiotics in a clear reference to the ability of these molds to adapt to the ocean up this adaptation to the occurrence of genetic mutations Genetic diversity and relationships were studied using the RAPD technique. It produced four primers (111) major pandas, including 9 unique pandas, common pandas (14) and polymorphic forests. Inflated ranges ranged from 100 to 2,000 basis points. The value of the genetic polymorphism of each primer ranged between 33-100%. In terms of unique band patterns, the most characteristic band pattern for the number isolated with the primer OP-M06 and OP-R06 was given 37 pandas of which 3 were unique in each. Genetic distances ranged from 0.22805 to 0.66905 between A. fumigatus isolates. Cluster analyzes were conducted to construct a dendrogram tree showing the interconnection of isolates. Most of the isolated A. fumigatus isolates from the patient developed come from the same region (conservative) in close relationship (sub-mass) indicating a relationship between RAPD patterns and origin of isolates.

Virulence Determinants of Buffalo Mastitis Originated Streptococcus agalactiae Isolates

Streptococci as a cause of mastitis have become the major concern to the dairy industry worldwide due to huge economic losses. Streptococcus agalactiae is a major contagious mastitis pathogen and continues to be a major cause of mastitis in dairy cattle and buffaloes. The aim of the study was to investigate the virulence determinants of S. agalactiae strains isolated from buffalo milk. Within the scope of the study, 24 S. agalactiae isolates from buffalo mastitis were examined. Biofilm production of isolates was investigated phenotypically by CRA method. It was observed that 18 (75%) isolates were positive for biofilm production. The presence of hylB, fnbB, scpB and spb1 virulence genes in S. agalactiae isolates were investigated by PCR. It was determined that 19 (79.17%) of the isolates were positive for scpB and 6 (25%) for fnbB virulence genes. None of the isolates were found to contain hylB and spb1 virulence genes. The antibiotic resistance profiles of the isolates among kanamycin, ampicillin, enrofloxacin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole antibiotic discs were determined by Kirby Bauer Disc Diffusion Method. Resistance were evaluated as % 41.7, % 45.9, % 25, % 12.5, % 20.9, and % 33.3, respectively. RAPD-PCR patterns of all isolates were determined using the ERIC-2 primer. The dendrograms of the RAPD patterns were plotted with the UPGMA method. It was determined that the isolates showed similarity between 59-95%. In conclusion, the research confirms the prevalence of various virulence genes in S. agalactiae isolated from buffalo mastitis. Further studies are therefore necessary to determine the molecular epidemiology and variability of S. agalactiae isolated from buffaloes, with the aim of improving mastitis control programs with regard to S. agalactiae.

Virulence Gene Properties and Phylogenetic Relationship of Salmonella Serotypes Isolated from Different Sources in North of Iran

Background: Salmonella species can cause various infections in humans and animals. The presence of certain genes determines the virulence of a Salmonella serotype. Objectives: The current research endeavor was undertaken to assess the virulence characteristics and genotypic traits of Salmonella serotypes extracted from various sources within the geographical boundaries of Iran. Methods: Salmonella isolates, previously retrieved and preserved in the veterinary microbiology laboratory, underwent serotyping and polymerase chain reaction (PCR) identification of nine virulence-associated genes. Genotyping was carried out using random amplified polymorphic DNA-PCR (RAPD-PCR). Results: All Salmonella isolates showed the presence of invA, sdiA, hilA, and iroB virulence genes. There were a total of 17 different virulence gene patterns among Salmonella serotypes. The presence of fliC and sefA genes and their related patterns were significant among S. typhimurium and S. enteritidis serotypes, respectively (P < 0.05). In the RAPD-PCR fingerprinting, 11 distinct clusters were obtained, and 16 isolates (26.66%) were classified as untypeable strains. There was a significant association between RAPD genotypes and Salmonella serotypes (P < 0.05), while the association between these RAPD patterns and the source of the isolates was not significant (P > 0.05). Conclusions: According to the results, Salmonella serotypes from non-human sources carry significant virulence determinants and show similar genotypic patterns with human isolates. These findings provide valuable insights into the virulence properties and genetic diversity of Salmonella serotypes in Iran, which could inform the development of effective control and prevention strategies for salmonellosis in the region.

First Report of Pectobacterium carotovorum and Pectobacterium aroidearum Causing Bacterial Soft Rot on Curly Dwarf Pak Choy (Brassica rapa var. Chinensis) in Malaysia.

Brassica rapa var. Chinensis (curly dwarf pak choy) is commonly grown in large-scale vertical farming aquaponic systems. In October 2022, soft rot symptoms and dark brown lesions were observed on B. rapa grown in a commercial aquaponic farm located in Perak, Malaysia. The infected stem appeared brown and water soaked. Severely infected plants produced creamy white ooze on the surface before collapsing entirely (Fig. 1A and B). Infected leaves displayed yellow-brown symptoms and eventually rotted (Fig. 1C); the healthy plants were symptomless (Fig. 1D). About 20 % of the 20,000 B. rapa plants on the farm exhibited symptoms. Ten randomly selected symptomatic plants, five with infected stems and five with infected leaves, were surface sterilized. Each tissue (1.0 cm2) was homogenized and suspended in a saline solution. The suspensions were then serially diluted and plated separately on Luria-Bertani agar. After a 16-h incubation period, stem tissue yielded 12 isolated colonies, while leaf tissue produced 8 colonies. These isolates were subjected to dereplication using RAPD-PCR (Krzewinski et al., 2001), revealing two distinct RAPD patterns. The cultures, named Pathogen Stem 2 (PS2, obtained from the stem) and Pathogen Leaf 2 (PL2, obtained from the leaf), were initially identified as Pectobacterium sp. through 16S rRNA sequence analysis (Frank et al., 2008) on the EzBioCloud 16S database (Yoon et al., 2017). Further identification of the Pectobacterium species was conducted using multilocus sequence analysis (MLSA) of the icdA, mdh, proA, and mltD genes (Ma et al., 2007). The sequences were deposited in GenBank (OQ660180, OQ660181, and OR206482-OR206489). Based on MLSA phylogeny, PS2 and PL2 were identified as Pectobacterium carotovorum and Pectobacterium aroidearum, respectively (Fig. 2A). Anaerobic assays confirmed their facultative anaerobic nature, while Gram staining revealed Gram-negative, rod-shaped morphology consistent with Pectobacterium (Fig. 2B and C). For the re-inoculation study, one-month-old healthy B. rapa plants were used. PS2 was inoculated into petioles, while PL2 was inoculated into leaves separately (3 biological replicates × 3 leaves for each replicate) using the prick inoculation method (Wei et al., 2019). Sterile needles were used to prick the plant tissues, and 10 µL of bacterial suspensions (2.40×109 CFU/mL) in saline were inoculated onto the pricked spots. Negative control using sterile saline was included. The inoculated plants were maintained in a controlled growth chamber (25 ± 1°C, relative humidity 80 ± 5%). After 48 hpi, the petiole tissue inoculated with PS2 showed bacterial soft rot symptoms (Fig. 1F) and leaves inoculated with PL2 appeared dark brown around the wound (Fig. 1G), similar to the symptoms observed in the commercial farm (Fig. 1B, C); while control plants remained asymptomatic (Fig. 1E). Bacteria were re-isolated from the inoculated petiole and leaf tissue and their identities were confirmed by RAPD-PCR. The RAPD profiles of the bacteria reisolated from the petiole and leaf tissues were the same as those of PS2 and PL2 respectively (Fig. 1H). The pathogenicity of PS2 and PL2 was thus confirmed. To our knowledge, this is the first report of bacterial soft rot on B. rapa in aquaponic systems caused by P. carotovorum and P. aroidearum in Malaysia. The identification of these pathogens is crucial for the prevention of disease outbreaks and to develop an effective disease management strategy.

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.

Assessment of the genetic fidelity of true-to-type regenerants of medicinal plant Rheum emodi using RAPD and ISSR molecular markers

Rheum emodi commonly known as rhubarb is mainly found in Northern Himalayas. It is a valuable medicinal plant having major pharmacological activities such as antimicrobial, anticancer, antioxidant, anti-inflammatory and is used extensively as purgative, stomachic and astringent tonic and improves gastro related problems. This herb is used by the local communities for medicinal as well as common eating purpose. This leads to its immense decline in its natural habitat and now this herb falls under threatened species and demands conservation. In this prospective, an efficient in vitro propagation method from callus culture has been achieved using leaf explants excised from the juvenile plant of R. emodi. Murashige and Skoog (MS) basal medium was used for regeneration procedure with different concentration of phytohormones. Maximum frequency of callus formation (84.44±0.27%) was observed in MS+36.19μM (2, 4-D) in combination with 11.10μM (BAP). The highest percentage of adventitious shoot regeneration was observed as 75.56±0.27% and the maximum number of shoots per explant that is 3.67±0.27 was achieved on MS basal medium containing BAP (35.5 μM) and Kn (11.61 μM). The maximum frequency of rooting was observed in MS full strength media + IAA (28.55 μM) + BAP (8.88 μM). The highest frequency of roots per shoot was observed as 5.0±0.47 with an average root length of 11±1.25mm. For ascertaining the clonal fidelity, 20 ISSR markers and 15 RAPD markers were assayed and employed to validate the true-to-type regenerants of Rheum emodi. Out of 15 RAPD and 20 ISSR markers, 7 markers and 15 markers produced distinct, clear and scorable bands with an average of 4.5 bands and 4.4 bands per marker respectively among the tissue cultured progenies. For each primer, the banding pattern was uniform and comparable to mother plant and showed about 99% homology. All the markers produce the monomorphic bands and no variation was detected among the micropropagated plants. Thus, the analysis of ISSR and RAPD patterns revealed that the bands were shared by both in vitro raised plants and parent clump confirming the genetic stability. DNA based molecular markers have proved to be versatile tools in diverse fields of biology. These markers proved to be model tools for routine analysis of clonal fidelity of micropropagated plants prior to commercialization.

Selection of Autochthonous Yeasts Isolated from the Intestinal Tracts of Cobia Fish (Rachycentron canadum) with Probiotic Potential

Some yeast strains have been proposed as probiotics to improve the health of cultured fish. Cobia is a tropical benthopelagic fish species with potential for marine aquaculture; however, one of the main limitations to its large-scale production is the high mortality of fish larvae. In this study, we evaluated the probiotic potential of autochthonous yeasts from the intestines of cobia. Thirty-nine yeast isolates were recovered from the intestinal mucosa of 37 adult healthy cobia by culture methods. Yeasts were identified by sequencing of the ITS and D1/D2 regions of the 28S rRNA gene and typed by RAPD-PCR using the M13 primer. Yeast strains with unique RAPD patterns were characterized in terms of their cell biomass production ability; anti-Vibrio, enzymatic, and hemolytic activity; biofilm production; hydrophobicity; autoaggregation; polyamine production; safety; and protection of cobia larvae against saline stress. Candida haemuloni C27 and Debaryomyces hansenii C10 and C28 were selected as potential probiotics. They did not affect the survival of larvae and showed biomass production >1 g L-1, hydrophobicity >41.47%, hemolytic activity γ, and activity in more than 8 hydrolytic enzymes. The results suggest that the selected yeast strains could be considered as potential probiotic candidates and should be evaluated in cobia larvae.

Clonal relatedness and antimicrobial susceptibility of Salmonella serovars isolated from humans and domestic animals in Iran: a one health perspective.

Salmonellosis is one of the most important zoonotic diseases in humans and animals worldwide. The main objective of this study was to report serovars, clonal relatedness, and antimicrobial resistance of Salmonella strains isolated from human, different animal hosts including pigeons, broilers, cattle, camel, parrots, and hamsters in different regions of Iran. Twenty-four Salmonella isolates were confirmed at the genus level by biochemical tests and polymerase chain reaction (PCR) by showing the presence of invA gene. Serovars were determined and their clonal relatedness was assessed by RAPD-PCR and antibiotic resistance profiles. Overall, Salmonella Typhimurium was the most prevalent serovar (45.8%, 11/24), which was recovered from humans, pigeons, and camels. Salmonella Enteritidis (29.2%, 7/24) was the second common serovar that was recovered from cattle, broilers, humans, and hamsters. Salmonella Infantis (12.5%, 3/24) belonged only to broiler sources, and Salmonella Seftenberg (12.5%, 3/24) was isolated from eggs and a parrot. The major RAPD pattern was VI (33.3%) in which the two S. Typhimurium isolates (belonged to humans and pigeons) exhibited similarity in both RAPD pattern and resistance profile. Antimicrobial susceptibility test showed full resistance to tylosin and erythromycin (100%, 24/24). All isolates (100%, 24/24) were susceptible to ceftriaxone, cefixime, and gentamicin. In total, 75% of the isolates were multi-drug resistant (MDR) and revealed 15 different antimicrobial resistance profiles (R-type). This study supports the potential transmission of Salmonella serovars via animal contacts. Thus, it is necessary to establish a national systematic monitoring program with one health approach for controlling Salmonella infections.

In vitro tesztrendszer alkalmazása probiotikus baktériumtörzsek szelektálására

The aim of our studies was to evaluate in vitro methods for the simple and efficient selection of putative probiotic bacterial strains. Of the possible methods, the following were tested: culturing on selective media, Gram staining, catalase assay, hemolytic, clonality and aggregation ability, gastric acid tolerance and bile acid tolerance. A total of 217 bacterial strains isolated from raw sheep’s milk, curdled milk and sheep’s cheese samples produced in Transylvania were included in our experiments. Isolates with hemolytic activity, as well as those exhibiting Gram-negative or catalase-positive phenotypes not characteristic of probiotics were excluded from our studies. Based on the results of RAPD-PCR studies suitable for the detection of individual-level polymorphisms, a total of 34 clone classes and 57 strains with unique RAPD patterns were identified. From each of the 34 clone classes thus narrowed, one strain was selected and tested for its aggregation ability, as well as its gastric acid and bile acid tolerance. High aggregation values above 70%, typical of probiotic strains, were measured in the case of a total of six isolates. In the course of the presence-absence studies conducted on the surface of solid media supplemented with acid or bile acid, it was possible to select several strains specifically tolerant to acid or bile acid. Based on our results, isolates to be included in further tests, e.g., in antibiotic resistance and antimicrobial activity assays, were selected.

The conservation strategy of a short lifespan and small genet fungus after thinning

Thinning removes a fraction of trees within a stand and as a result, forest fungi faces threats, such as habitat loss and fragmentation. After thinning, a reduction in sporocarp production of small and isolated fungal populations may be particularly worrisome from a conservation perspective.A clavarioid fungus, Scytinopogonsp., was sensitive to thinning and provided an ideal model to investigate how the fungus reproduces in nature and how the population copes with constantly changing environments. Here, we investigated its genetic variations by RAPD and monitored its population dynamics for 7 year. Results of DNA fingerprinting shows that Scytinopogonsp. population consists of numerous small and short-lived genets, which suggested that sexual reproduction and recombination are common and important forScytinopogonsp. population. The Scytinopogon sp. population was declined after thinning and started to increase and recover in the third year after thinning. Genetic differentiation in Scytinopogon sp. was observed after thinning. The recovered subpopulation in thinned plots showed similar RAPD patterns as the subpopulations from nearby plots of other treatments in the same year. That suggested recovery of the basidiomata is established by the basidiospores recruited from the adjacent un-thinned area and 25% thinned plots, while the original subpopulations went locally extinct in heavily thinned plots. To conserve rare fungal species, reduction of disturbance, conservation of habitat and protection of the minimum viable population have to be considered during forest management.

Changes in RAPD-DNA Markers and Plasma Protein Profile in Progesterone-Treated Chicken

The abuse of contraceptive pills as growth promoters in the poultry industry has serious health concerns. Consumption of hormonal residues in animal products results in reproductive disorders and an increased risk of cancers in humans. This study aimed to investigate the effect of treatment with progesterone hormone on the plasma proteins and RAPD-DNA markers of broiler chickens. One hundred, one-day-old broiler chicks were equally divided into 5 groups (n = 20). Four groups were treated daily with progesterone hormone either subcutaneously or orally (1.0 mg, 1.5mg/kg). The 5th group was control non-treated chicken. Sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE), and RAPD assays in conjunction with productive parameters such as growth rate were assessed. SDS-PAGE and RAPD-DNA patterns were significantly affected by progesterone hormone treatment. Either or oral or subcutaneous progesterone hormone treatments increased the body weight of chicks compared to control. Treatment with progesterone hormone induces effects on both DNA and plasma proteins in broiler chickens. Changes at the genomic DNA level may be due to specific modifications in RAPD patterns or arise as a consequence of hot spot DNA changes and/or mutations.

Identification of Four Molokhia (Corchorus olitorius L.) Genotypes by Molecular Markers

Assessment of genetic diversity in a collection of genetic resources is a vital characterization for preserving this collection and planning future breeding programs. Genetic variability among four Egyptian molokhia (Corchorus olitorius L.) genotypes was observed using random amplified polymorphic (RAPD) DNA. RAPD patterns with polymorphism percentages were detected among the DNA samples from seven-day-old seedlings, representing each of the studied genotypes. Genetic diversity and relationships were evaluated using five randomly amplified polymorphic DNA markers that revealed 288 scorable bands. Amplification products were ranged in size from 110 bp to 5.5 kb, while in number, they ranged from 2 to 17 bands. Indicated significant genetic diversity among the molokhia genotypes were 66.67% for Wild type x Balady, 65.28% for Wild type x Saidy, 63.39% for Wild type x Falahy. The closest proximity, incidentally the highest genetic similarity, was observed between Balady and Falahy (80.56%), while the lowest was observed between Balady and Saidy (56.94%). The total positive unique bands scored 11 bands, while the negative unique bands scored 8 within the tested genotype and the five primers. Studied five primers generated unique bands in three molokhia landraces, which could identify these genotypes at the molecular level. The results suggest considerable potential RAPD approach for proper genetic identification of individual genotypes and an efficient way of varietal authenticity for fraud prevention. Genetic variability within molokhia genotypes ascertained using these primers. Results recommended the usefulness of RAPD primers in conservation and exploitation of molokhia germplasm in wide variety of breeding programs.

Sex identification in Nepenthes adrianii from Baturraden Botanical Garden: Genetic analysis using RAPD Markers

Nepenthes adrianii is one of pitcher plant species that grows endemically in Mount Slamet, Central Java. At present, it is one of pitcher plant collections of Baturraden Botanical Garden. Since N. adrianii is dioecious and both sexes are difficult to distinguish morphologically, early sex determination supporting its conservation at Baturraden Botanical Garden is needed. One approach can be performed with the use of RAPD molecular markers. Hence, this study aims to know whether differences in RAPD pattern between male and female N. adrianii exist or not and also to find out what the differences are. Genomic DNAs were extracted from leaves of 4 males, 2 females, and 2 sexually unidentified individuals. The extracted DNAs were then used to analyze DNA variation between male and female N. adrianii employing RAPD technique. As many as five oligonucleotide primers (OPA-15, OPK-16, OPP-15, OPP-08, and OPO-08) were used to amplify N. adrianii DNA. The results showed that one primer, i.e. OPK-16 (5’-GAGCGTCGAA-3’), produces a specific band of approximately 290 bp which is only found in female plants. It is assumed that this band is related to gene(s) controlling sex determination in N. adrianii. The RAPD marker can be used for the sex determination of young N. adrianii seedlings.

Real-Time PCR Assays for Rapid Identification of Common Aphanomyces astaci Genotypes

The oomyceteAphanomyces astaciis the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. TrackingA. astacigenotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups ofA. astaci(A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pureA. astacicultures representing the known genotype groups, and fromA. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded byA. astaci(particularly Europe). The unusualA. astacigenotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infectedPontastacus leptodactylusin the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity withAphanomyces fennicus; theA. astacigenotype “rust1” described in the United States fromFaxonius rusticusis detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping ofA. astacigenotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.

High frequency of blaPER-1 gene in clinical strains of Acinetobacter baumannii and its association with quorum sensing and virulence factors

Acinetobacter baumannii is known as the main pathogenic organisms, especially for the nosocomial infections. Multi-drug resistance and the presence of different virulence factors are recognized as causes of the organism's survival and the infection's risk. In this study, 70 Acinetobacter baumannii isolates were collected from three hospitals located in the Northwest of Iran. Antimicrobial susceptibility of these isolates was determined by agar dilution and disk diffusion methods. Production of gelatinase, proteolytic, and hemolytic activities of the isolates was also assessed. The frequencies of epsA, pld, ptk, luxR, luxI, and blaPER-1 genes were investigated by polymerase chain reaction (PCR). Furthermore, molecular typing of the isolates was performed using random amplified polymorphic DNA (RAPD-PCR). All the obtained isolates were multi drug resistant and 58.5% of them were extensively drug resistance. Six of the 70 study isolates were found to be resistant to all the tested antibiotics. Nine (12.8%) of the isolates were found to be resistant to colistin (MIC = 4 μg/mL). 38.5%, 45.7%, and 15.5% of the isolates were positive for gelatinase production, protease, and hemolysis, respectively. The frequency of the genes was as follows: pld 100%, epsA 91.9%, luxI 85.7%, ptk 80%, luxR 75.7% and blaPER-1 64.2%. Based on the RAPD patterns obtained, the 70 isolates in this study clustered into twelve major genotypes. In this study, the prevalence of blaPER-1 was worryingly high. On the other hand, a significant relationship was found between the presence of this gene and the production of some virulence factors in the strains.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of β-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

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.

Genotyping by Random Amplified Polymorphic DNA Assay of Acinetobacter baumannii Isolated from Blood Culture of Patients with Hematological Malignancies

Relevance. Acinetobacter baumannii is a significant nosocomial pathogen that can cause severe infections, especially in immunocompromised patients. Aims. This study aimed to investigate clonal diversity of A. baumannii isolated from blood culture in hematological patients by random amplified polymorphic DNA assay (RAPD). Materials & Methods. Genotyping of A.baumannii isolated from blood culture in hematological patients in 7 Russian hospitals (2003–2017) was assessed by RAPD-PCR with primer OPA-2 (5’-TGCCGAGCTG-3’). The computer-assisted analysis was performed by using GelJ software by UPGMA method and Dice similarity coefficient for banding patterns comparison. Using a similarity coefficient (SC) of ≥ 65%, the strains were grouped. Based on the similarity coefficient, the strains were determined as genetically related (≥ 80%). Strains had identical RAPD-patterns if the similarity coefficient was 100%. Results. A total of 96 A. baumannii strains were examined, of those 77 (80.2%) were nonsusceptible to carbapenems. Acquired OXA-carbapenemase genes were detected among 79.2% carbapenem non-susceptible strains. RAPD-PCR genotyping revealed 84 RAPD patterns. The four groups (A-D) including 98% strains were defined by similarity coefficient ≥65%. The predominant group A included 58 (60.4%) strains, the C and B groups – had 15 strains (15.6%) each, and the group D – 6 strains (6.3%). A total of 82 (85.4%) genetically related A. baumannii with a similarity coefficient ≥ of 80% were allocated into 20 clusters. Identical RAPD-patterns were defined for 22 strains that belonged to 6 clusters within the group A and 1 cluster within the group B. Strains with identical RAPD-patterns were detected in a single hospital as well as in several hospitals located in different cities. Conclusions. The current study has demonstrated genetic diversity and clonal dissemination of A. baumannii in hematological departments.

High counts of thermophilic spore formers in dairy powders originate from persisting strains in processing lines

During the last decades, thermophilic spore counts became a very important quality parameter for manufacturers with regard to powdered dairy products. Low-spore count powders are highly demanded but challenging to produce when high production volume and long process times are intended. In this study a detailed monitoring of microbial levels in three skim milk powder plants was conducted. Anoxybacillus flavithermus was found to be primarily responsible for increased spore levels with increasing spore numbers being detected after 6–8 h already during initial processing steps. Simultaneously, the species composition shifted from a diverse bulk tank milk microbiota where different Bacillus species represented around 90% of the thermophilic bacteria to a dominance of A. flavithermus in the end product. The analysis of A. flavithermus isolates from different powder batches with RAPD PCR revealed recurring patterns in each of the eight German manufacturers sampled over several months. The high relatedness of isolates exhibiting identical RAPD patterns was exemplified by cgMLST based on whole genome sequences. We assume that A. flavithermus strains persisted in production plants and were not eliminated by cleaning. It is concluded that such persisting strains recurrently recontaminated subsequent powder productions. The data highlight that a targeted optimization of cleaning and disinfection procedures is the most promising measure to effectively reduce thermophilic spore counts in German dairy powders.

Screening and identification of bacteriocin-like inhibitory substances producing lactic acid bacteria from fermented products

Abstract A total of 544 isolates of lactic acid bacteria isolated from fermented products: 451 isolates from fermented products from Thailand and 93 isolated from French fermented products were screened for bacteriocin production. 10 isolates showed antimicrobial activities. Identification of the selected strains by random amplification of polymorphic DNA-polymerase change reaction using the primers E1, E2 and M13 showed three different RAPD patterns. One of the isolates from each product was chosen and good antimicrobial activity were displayed by 3 strains including MP6/2, PKS2-1 and OV1-1 producing in MRS broth bacteriocin-like substance (BLIS) inhibiting Candida albicans ATCC 90028, Carnobacterium maltraromaticum NCDO 2760, Lactobacillus sakei subsp. sakei JCM 1157, Listeria innocula CIP 80.11, Listeria ivanovii SLCC 2379, Pseudomonasc aeruginosa ATCC 27853, Staphylococcus aureus CIP 76.25 and Streptococcus mutans used as indicator strains. However, inhibitory activities of MP6/2 and PKS2-1 were related to the production of either organic acid, while OV2-1 was not affected by heating. The inhibitory activities of these three isolates were sensitive to proteolytic enzymes. 16S rDNA gene sequencing allowed to identify the MP6/2 as Pediococcus pentosaceus while PKS2-1 and OV1-1 were identified as Enterococcus faecium. It was concluded that the obtained isolates may be used as bio-preservative cultures to produce fermented foods.