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Abstract This is the primary study at Matrouh Governorate to unveil antibiotic resistance, biofilm formation, silver nanoparticles (Ag-NPs) effect using electron microscopy, and REP-PCR analysis of Staphylococcus aureus strains isolated from COVID-19 patients, contaminated food, and Morel’s diseased sheep and goats. A total of 15 S. aureus strains were isolated; five from each of the COVID-19 patients, Morel's diseased sheep and goats, and contaminated food. All strains were considered multidrug-resistant (MDR). All strains showed the presence of biofilm. Morphological changes in the cell surface of the bacterium were evidenced, and penetration with the rupture of some bacterial cells. Based on REP-PCR analysis, 4 clusters (C1-C4) with dissimilarity between clusters C1 and C2 8% and between C3 and C4 15%. Cluster I included 3 strains from contaminated food with a similarity of 97%, and Cluster II included 2 strains from contaminated food and 2 from COVID-19-infected patients with a similarity of 96% (confirming the zoonotic nature of this pathogen). Cluster III contained 4 strains isolated from Morel's diseased sheep & goats with a similarity ratio of 99% in comparison the 4th cluster contained 3 strains isolated from COVID-patients and one from Morel's diseased sheep & goats with a similarity ratio of 92%.

This is the primary study at Matrouh Governorate to unveil antibiotic resistance, biofilm formation, silver nanoparticles (Ag-NPs) effect using electron microscopy, and REP-PCR analysis of Staphylococcus aureus strains isolated from COVID-19 patients, contaminated food, and Morel's diseased sheep and goats. A total of 15 S. aureus strains were isolated; five from each of the COVID-19 patients, Morel's diseased sheep and goats, and contaminated food. All strains were considered multidrug-resistant (MDR). All strains showed the presence of biofilm. Morphological changes in the cell surface of the bacterium were evidenced, and penetration with the rupture of some bacterial cells. Based on REP-PCR analysis, 4 clusters (C1-C4) with dissimilarity between clusters C1 and C2 8% and between C3 and C4 15%. Cluster I included 3 strains from contaminated food with a similarity of 97%, and Cluster II included 2 strains from contaminated food and 2 from COVID-19-infected patients with a similarity of 96% (confirming the zoonotic nature of this pathogen). Cluster III contained 4 strains isolated from Morel's diseased sheep & goats with a similarity ratio of 99% in comparison the 4th cluster contained 3 strains isolated from COVID-patients and one from Morel's diseased sheep & goats with a similarity ratio of 92%. [ABSTRACT FROM AUTHOR]

Escherichia coli is one of the most important pathogens causing bloodstream infections (BSIs) throughout the world. We sought to characterize the phylogroup classification, major human sequence types (STs), antimicrobial resistance, presence of selected antimicrobial resistance and virulence genes, and genetic diversity of E. coli isolated from patients with BSIs at the University Hospital in Iran. A total of 100 E. coli bloodstream isolates were collected between December 2020 and June 2022. This study used PCR to investigate phylogenetic groups (A, B1, B2, C, D, E, and F), four major STs (ST69, ST73, ST95, and ST131), antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), and pathogenicity islands (PAIs). Antimicrobial susceptibility testing was done by disk diffusion method. Genetic diversity was analyzed by repetitive element sequence-based PCR (REP-PCR). The phylogenetic group B2 (32%) predominated, followed by phylogenetic group E (25%). ST131 (28%) was the most prevalent ST and the majority of these isolates (89.3%) were of serotype O25b. Most of E. coli isolates (75%) were categorized as multidrug resistant (MDR) with high rates of resistance (>55%) to ampicillin, trimethoprimsulfamethoxazole, ciprofloxacin, cefazolin, and ceftriaxone. The most frequent ARGs were blaTEM (66%), sul1 (57%), and sul2 (51%). The most prevalent VAGs and PAIs were fimH (type 1 fimbriae adhesin; 85%), aer (iucC) (aerobactin; 79%), traT (serum resistance; 77%), iutA (aerobactin siderophore receptor; 69%), and PAI IV536 (75%), respectively. The highest rate of ARGs and VAGs was observed in the ST131 isolates. REP-PCR analysis showed high diversity among the studied isolates. The high prevalence of MDR septicemic E. coli with different types of ARGs, VAGs and genotypes is an extremely worrisome sign of BSIs treatment and poses a major threat for hospitalized patients. Active surveillance, stringent prescribing policies, increasing the awareness of ARGs among clinicians and redefining the infection control measures are essential to curb the dissemination of these strains. [ABSTRACT FROM AUTHOR]

Seventy-five pectinolytic strains collected from vegetables grown in the counties of Santarém, Belterra, and Mojuí dos Campos, located in the western region of the Pará State, Brazil, were studied according to their pathological and genetic variability. The strains were grouped in 5 clusters according to pathogenicity in potato, pepper, carrot, and onion, and 38 strains were selected for genetic analysis using rep-PCR. These strains were divided into 35 genetic groups according to rep-PCR at 70% similarity. These results indicated high pathological and genetic variability of the strains causing soft rot in vegetables in the western region of the Pará State, which will be used in etiological research and for the development and assessment of management techniques for the soft rot in vegetables in this region. [ABSTRACT FROM AUTHOR]

Abstract Background The phylogenetic relationships and phylogeny of twenty-six Trichoderma species collected from various Egyptian locations were investigated. The genetic diversity among the examined isolates was tested using the rep-PCR marker. Trichoderma species were screened for their cellulase activities. Results Three isolates demonstrated highly significant FPase activities, namely MNF-MAS-Tricho 1, MNF-MAS-Tricho 2, and MNF-MAS-Tricho 3 (0.50, 0.39, and 0.49 IU ml−1, respectively). MNF-MAS-Tricho1 showed the highest significant CMCase activity (0.80 IU ml−1). Concerning β-glucosidase, MNF-MAS-Tricho 1 was the highest (0.78 IU ml−1), while MNF-MSH-Trich 11 and MNF-MAS-Tricho 15 were the lowest (0.36 IU mL−1). The percentage of polymorphism ranged from 46.15 to 83.33%. (GTG)5 marker produced the greatest number of polymorphic loci (13 loci out of 18 loci) with about 83.33% polymorphism, followed by rep-10 with 69.2% polymorphism. Furthermore, the polymorphism information content (PIC) estimates ranged between 0.285 for Rep-10 and 0.340 for (GTG) 5 with an average of 0.306. The tested primers exhibited high discriminating and resolving powers. Conclusion The findings of this investigation were used to classify Trichoderma species, evaluate their genetic variability using ITS sequencing, rep-PCR, and measure their cellulase activities. These markers can facilitate more rapid and less complicated studies of Trichoderma population dynamics and evaluate their establishment after release into agricultural environments. The results will help to evaluate the genetic diversity of Trichoderma in future research.

Background: The phylogenetic relationships and phylogeny of twenty-six Trichoderma species collected from various Egyptian locations were investigated. The genetic diversity among the examined isolates was tested using the rep-PCR marker. Trichoderma species were screened for their cellulase activities. Results: Three isolates demonstrated highly significant FPase activities, namely MNF-MAS-Tricho 1, MNF-MAS-Tricho 2, and MNF-MAS-Tricho 3 (0.50, 0.39, and 0.49 IU ml−1, respectively). MNF-MAS-Tricho1 showed the highest significant CMCase activity (0.80 IU ml−1). Concerning β-glucosidase, MNF-MAS-Tricho 1 was the highest (0.78 IU ml−1), while MNF-MSH-Trich 11 and MNF-MAS-Tricho 15 were the lowest (0.36 IU mL−1). The percentage of polymorphism ranged from 46.15 to 83.33%. (GTG)5 marker produced the greatest number of polymorphic loci (13 loci out of 18 loci) with about 83.33% polymorphism, followed by rep-10 with 69.2% polymorphism. Furthermore, the polymorphism information content (PIC) estimates ranged between 0.285 for Rep-10 and 0.340 for (GTG) 5 with an average of 0.306. The tested primers exhibited high discriminating and resolving powers. Conclusion: The findings of this investigation were used to classify Trichoderma species, evaluate their genetic variability using ITS sequencing, rep-PCR, and measure their cellulase activities. These markers can facilitate more rapid and less complicated studies of Trichoderma population dynamics and evaluate their establishment after release into agricultural environments. The results will help to evaluate the genetic diversity of Trichoderma in future research. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli is the most common etiological agent of urinary tract infections (UTIs). Meanwhile, plasmid-mediated quinolone resistance (PMQR) is reported in E. coli isolates producing extended-spectrum β-lactamases (ESBLs). Furthermore, the reservoirs and mechanisms of acquisition of uropathogenic Escherichia coli (UPEC) strains are poorly understood. On the other hand, UTIs are common in pregnant women and the treatment challenge is alarming. Methods and results: In the present study, 54 pregnant women with acute cystitis were included. A total of 108 E. coli isolates, 54 isolates from UTI and 54 isolates from faeces of pregnant women (same host) were collected. In the antimicrobial susceptibility test, the highest rate of antibiotic resistance was to nalidixic acid (77%, 83/108) and the lowest rate was to imipenem (9%, 10/108). Among the isolates, 44% (48/108) were ESBLs producers. A high frequency of PMQR genes was observed in the isolates. The frequency of PMQR genes qnrS, qnrB, aac(6′)-Ib-cr, and qnrA was 58% (63/108), 21% (23/108), 9% (10/108), and 4% (4/108), respectively. Meanwhile, PMQR genes were not detected in 24% (20/85) of isolates resistant to nalidixic acid and/or fluoroquinolone, indicating that other mechanisms, i.e. chromosomal mutations, are involved in resistance to quinolones, which were not detected in the present study. In ESBL-producing isolates, the frequency of PMQR genes was higher than that of non-ESBL-producing isolates (81% vs. 53%). Meanwhile, UTI and faeces isolates mainly belonged to phylogenetic group B2 (36/54, 67% and 25/54, 46%, respectively) compared to other phylogenetic groups. In addition, virulence factors and multidrug-resistant (MDR) were mainly associated with phylogenetic group B2. However, predominant clones in faeces were not found in UTIs. Rep-PCR revealed the presence of 85 clones in patients. Among the clones, 40 clones were detected only in faeces (faeces-only), 35 clones only in UTI (UTI-only) and 10 clones in both faeces and UTI (faeces-UTI). We found that out of 10 faeces-UTI clones, 5 clones were present in the host's faeces flora. Conclusion: This study revealed a high rate of resistance to the quinolone nalidixic acid and a widespread distribution of PMQR genes in MDR E. coli strains producing ESBLs. The strains represented virulence factors and phylogenetic group B2 are closely associated with abundance in UTI and faeces. However, the predominant clones in faeces were not found in UTIs and it is possible that rep-PCR is not sufficiently discriminating clones. [ABSTRACT FROM AUTHOR]

Outbreaks of bacterial leaf blight (BLB) frequently affect eucalypt plants under nursery and field conditions in several countries. Although research has been conducted to unveil the causal agent, different bacterial species have been associated with similar disease symptoms in different countries. In order to determine the causal agent of BLB in Brazil, a survey was conducted in nine states to recover bacterial isolates from eucalypt plants exhibiting typical BLB symptoms. A total of 41 yellow‐colony isolates with varying aggressiveness towards a susceptible eucalypt clone were obtained, with 16S rDNA sequences indicating that they belong to the Xanthomonas genus. Rep‐PCR analysis separated the Xanthomonas population affecting eucalypt into six distinct groups revealing its high genetic diversity. The same population formed three clusters together with reference strains of X. citri, X. euvesicatoria and X. phaseoli in a phylogenetic tree constructed with partial dnaK, fyuA, gyrB and rpoD gene sequences. Clustering in the phylogenetic tree was clearly related to grouping based on rep‐PCR. Genome sequence comparisons of representative eucalypt isolates with type strains of validly published Xanthomonas species confirmed that the population consisted of X. citri, X. euvesicatoria and X. phaseoli. Inoculation of tomato, common bean, castor bean and eucalypt plants showed that the representative eucalypt isolates can cause disease in these plant species. Based on the results, the transfer of Xanthomonas axonopodis pv. eucalyptorum to Xanthomonas citri is proposed. These results are relevant for eucalypt BLB management under nursery and field conditions, including selection and deployment of effective plant resistance. [ABSTRACT FROM AUTHOR]

This study was conducted for identifying phylogenetic relationships between 15 scab-causing Streptomyces species including S. bottropensis, S. europaeiscabiei, S. scabiei, S. stelliscabiei and, other 11 Streptomyces sp. All of the strains were originally isolated from symptomatic potatoes in Erzurum Province, The Eastern Anatolia Region of Turkey. Some morphological and biochemical properties of the strains were defined in our former research. Then, 16 s rRNA regions of them were sequenced. After the sequence data assembly, phylogenetic analyzes were performed. The phylogenetic analyses revealed that the strains are involved in the same major group and, substantially similar to reference strains. Additionally, some subgroup formations were also recorded. Moreover, Repetitive element-based PCR (Rep-PCR), Enterobacterial repetitive intergenic consensus (ERIC-PCR), and BOX-PCR fingerprinting molecular typing methods were used for as molecular typing methods. According to our knowledge, this is the first report on phylogenetic relationships of scab-causing Streptomyces species from Turkey. However, the identification of most pathogenic strains remained at the species level. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus plantarum is widely distributed in nature and is one of the most widely developed and used lactic acid bacteria species at present. Most developed and used L. plantarum are derived from fermented food or plant-based materials. In this study, L. plantarum was isolated from fecal samples of healthy Uyghur and Kazakh children in Yining, Xinjiang, and 20 representative strains were selected by fingerprinting to remove the identical bands. The clustering results of fingerprinting classified the strains into three major groups, and strains of the same ethnicity and age group were more likely to be clustered in the same small branch. The phylogenetic tree based on groEL gene showed that strains of the same ethnic origin tended to cluster together. At the same time, the acid and bile salt tolerance, self-aggregation, hydrophobicity (xylene), bacteriostatic capacity, antibiotic sensitivity and carbohydrate utilization ability of 20 strains were tested in vitro. The results showed that strains YLW2L-108-29, YLW2L-61-7 and YLW1L-44-7 had better acid (pH2.5) resistance, and YLW1L-44-6 had the best bile salt tolerance (0.3% concentration). Strains YLW2L-61-7, YLW2L-57-4, YLW2L-66-30 had the best self-aggregation and hydrophobics results, and all strains grew well on fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, maltodextrin, raffinose, D-(+)-trehalose dihydrate, pectin, stachyose tetrahydrate and soybean oligosaccharide. Most strains had poor growth on inulin and resistant starch, and worst growth on xylo-oligosaccharide. Combined with the results of bacterial inhibition and antibiotic resistance, strain YLW2L-61-7 was screened as a potential probiotic strain for subsequent studies, laying the foundation for the development of excellent probiotic strains and products suitable to regional populations.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolations have rapidly increased in pediatric patients. To investigate a possible health care-associated infections of CRKP in a tertiary pediatric hospital, the circulating clones and carbapenem-resistant pattern between CRKP and carbapenem-resistant Acinetobacter baumannii (CRAB) isolates were compared to classify their epidemiological characteristics. The results will help to identify the epidemic pattern of the CRKP transmission in the hospital. Methods: Ninety-six CRKP and forty-eight CRAB isolates were collected in Kunming Children's Hospital from 2019 through 2022. These isolates were genotyped using repetitive extragenic palindromic-PCR (REP-PCR). Carbapenemase phenotypic and genetic characterization were investigated using a disk diffusion test and singleplex PCR, respectively. In addition, these characteristics of the two pathogens were compared. Conclusions: These CRKP isolates exhibited different biological and genetic characteristics with dynamic changes, suggesting widespread communities. Continuous epidemiological surveillance and multicenter research should be carried out to strengthen the prevention and control of infections. [ABSTRACT FROM AUTHOR]

ABSTRACT: Seventy-five pectinolytic strains collected from vegetables grown in the counties of Santarém, Belterra, and Mojuí dos Campos, located in the western region of the Pará State, Brazil, were studied according to their pathological and genetic variability. The strains were grouped in 5 clusters according to pathogenicity in potato, pepper, carrot, and onion, and 38 strains were selected for genetic analysis using rep-PCR. These strains were divided into 35 genetic groups according to rep-PCR at 70% similarity. These results indicated high pathological and genetic variability of the strains causing soft rot in vegetables in the western region of the Pará State, which will be used in etiological research and for the development and assessment of management techniques for the soft rot in vegetables in this region.

Phytophthora capsici and P. tropicalis are the two species of Phytophthora associated with foot rot disease of black pepper in India. High genetic diversity amongst the Phytophthora species contributes to its wide host range and variability in the virulence pattern. In the present study, genetic diversity of Phytophthora species infecting black pepper was analysed using RAMS (Random Amplified Microsatellites) and REP (Repetitive Extragenic Palindromic)-PCR fingerprinting. Forty-eight isolates, 24 each of P. capsici and P. tropicalis collected from major black pepper growing states, such as Karnataka, Kerala, Tamil Nadu and Goa, were used in the study. The analyses revealed a total of 160 loci of which 150 (93.75%) were polymorphic. UPGMA cluster and PCoA analysis based on combined RAMS and REP-PCR data clearly grouped the P. capsici and P. tropicalis isolates into two clusters which were further divided into four sub-clusters viz., I & II (P. capsici) and III & IV (P. tropicalis). The study clearly indicated that all the isolates were genetically unique and the entire population was heterogeneous. REP-PCR primers showed more polymorphic loci than RAMS primers. Further, sixteen isolates were selected for morphological and infectivity analyses under in vitro conditions. The isolates exhibited varied colony morphology, sporangial shapes and belonged to A1 mating type. Under in vitro conditions, all the sixteen black pepper Phytophthora isolates could infect nutmeg, tomato, chilli, pumpkin, and cucumber and few of the isolates could infect cardamom. None of the isolates could infect coconut, areca nut and vanilla. [ABSTRACT FROM AUTHOR]

Soft rot disease in orchid plants is a factor in decreasing orchid production. One way of biological control that has the potential to spur growth is to utilize endophytic bacteria. This study aims to describe the diversity of orchid endophytic bacteria that have the potential as growth promoters and biocontrol agents. Observation parameters include indole acetic acid (IAA) production, inorganic phosphate dissolution, ammonia production of orchid endophytic bacteria, and antagonism of endophytic bacteria to pathogenic bacteria that cause soft rot in vitro. The diversity of orchid endophytic bacteria was analyzed using rep-PCR with BOX-A1R primer. The results showed that 10 isolates of orchid endophytic bacteria were able to produce IAA with a concentration of 79–321 μg/mL, able to dissolve phosphate, and produce ammonia. In antagonism testing against soft rot-causing bacteria, all isolates were able to form an inhibitory zone with a diameter of 3–7.2 cm. Furthermore, diversity analysis using rep-PCR showed that isolates of endophytic bacteria DnPh5, BgCt2, BgVt10, DnLp7, DnBl1, and DnAr4 had a uniform DNA band pattern so that they were grouped together. Meanwhile, isolates of TbPh7, IbtPhm1, DnDr2, and AkOc1 showed different DNA band patterns, indicating that the four isolates were different species or subspecies of bacteria. In dendrogram analysis, DnPh5, BgCt2, BgVt10, DnLp7, DnBl1, and DnAr4 isolates have a similarity of 100%. The similarity of isolates IbtPhm1, DnDr2, and AkOc1 was 90%, 79%, and 68%, respectively, against the other six isolates. TbPh7 isolate is an isolate with a low level of similarity to other isolates, which is about 51%. [ABSTRACT FROM AUTHOR]

American foulbrood is an important bacterial disease affecting the larvae of honey bees (Apis mellifera L.) caused by Paenibacillus larvae. Due to easy transmission of disease and the ability of bacteria to create spores, it is a bacterium resistant to both physical and chemical conditions. The study aims to isolate, perform microbiological analyses, and determine biochemical properties and genotypes P. larvae strains from AFB samples collected from Turkey's Central and Eastern Black Sea regions. An isolation study was conducted on adult bees, larvae, honey, and primary honeycomb samples from suspected colonies in the regions under study. After the purification of bacterial isolates from samples, P. larvae strains were identified using biochemical and molecular methods. The genetic diversity and ERIC types of P. larvae isolates were determined by rep-PCR DNA genotyping using BOX A1R and MBO REP1 primers and multiplex-PCR. A phylogenetic tree of P. larvae strains was constructed in the study. All P. larvae isolates were determined as ERIC I type. According to the rep-PCR results of P. larvae strains, 15 of the 28 isolates were Ab genotype (54%), 7 (25%) Aβ genotype, 4 (14%) AB genotype, 1 (3.5%) αB genotype, and 1 (3.5%) ab genotype. From an epidemiological viewpoint, it was determined that Ab and Aβ genotypes were widely distributed, while other genotypes (AB, αB, and ab) showed less spread. The results of the study will guide researchers in taking relevant measures to prevent and control American foulbrood. [ABSTRACT FROM AUTHOR]

Objective Bacterial leaf streak (BLS) caused by of Xanthomonas translucens, is a serious bacterial seed-borne disease of wheat (Triticum aestivum L.) worldwide. This research was planned to study pathogenicity of the isolates obtained from bacterial leaf streak on wheat plants. Resistance of different wheat cultivars against the isolate with the highest pathogenicity was investigated. Molecular tools were used to identify bacterial isolates and their genetic diversity. Materials and methods Bacterial strains with yellow and soft colonies were isolated and their pathogenicity indicated that all selected isolates are pathogen. Aggressiveness of the isolates was differing among isolates. Wheats cultivars showed different responses to selected isolate, but all cultivars were susceptible to the pathogen. Result of molecular analysis with DNA fingerprinting techniques showed genetic diversity among the bacterial isolates obtained from different wheat growing regions. In the cluster analysis of the banding pattern obtained with ERIC and BOX primers, the isolates were divided into 5 main groups at 33% similarity. The results showed significant genetic diversity among isolates causing bacterial leaf streak disease in these provinces. Sequences comparison of gyrB and dnaK genes with similar sequences in the gene bank showed high homology with sequences from Xanthomonas translucens pv. undulosa. Results Bacterial strains with yellow and soft colonies were isolated and their pathogenicity indicated that all selected isolates are pathogen. Aggressiveness of the isolates was differing among isolates. Wheats cultivars showed different responses to selected isolate, but all cultivars were susceptible to the pathogen. Result of molecular analysis with DNA fingerprinting techniques showed genetic diversity among the bacterial isolates obtained from different wheat growing regions. In the cluster analysis of the banding pattern obtained with ERIC and BOX primers, the isolates were divided into 5 main groups at 33% similarity. The results showed significant genetic diversity among isolates causing bacterial leaf streak disease in these provinces. Sequences comparison of gyrB and dnaK genes with similar sequences in the gene bank showed high homology with sequences from Xanthomonas translucens pv. undulosa. Conclusions The rep-PCR techniques are suitable for studying genetic diversity of the obtained isolates. In this study, geographical location had no effect on the grouping of the isolates. Bacterial leaf streak disease is found in almost every wheat growing area and better understanding of the Xanthomonas translucens-wheat interactions are necessary to find and develop wheat cultivars with resistance to the disease.

In March-April of 2020-2022, round and water-soaked spots surrounded by a brown border were observed on the green bean pods delivered to the markets of Isfahan from the southern provinces of the country. A bacterium with a fluorescent appearance and a spindle-shaped structure with irregular edges was routinely isolated from the 81 specimens collected. The genetic fingerprinting employing rep-PCR revealed a resemblance between the PCR product profiles of the isolates, which is consistent with their pathogenic and biochemical characteristics. The PCR reaction of representative isolates produced the expected-size fragments of 16Sr DNA through genus-specific primer sets. Subsequent analysis of the sequences of the fragments confirmed the affiliation of the isolates with Pseudomonas spp. The expected-sized amplicons were amplified and sequenced in nested-PCR tests using phaseolotoxin gene based primer pairs. The results showed that the nucleotide sequences were 99% homologous to Pseudomonas savastanoi pv. phaseolicola (Psp). By conducting a phylogenetic analysis with sequences of housekeeping genes gyrB and rpoD, the isolates were clustered with reference strains of Psp, confirming their identity. Considering the isolates’ morphological, biochemical and molecular characteristics, Psp was identified as the cause of halo blight disease in beans imported to Isfahan from the southern regions. In laboratory conditions, the persistence of the pathogen on seed was evaluated for more than one year. The long-term viability of Psp on seeds may lead to the ongoing prevalence of bean halo blight disease in the country in the forthcoming years, due to the cool and moist conditions resulting from spring rains. [ABSTRACT FROM AUTHOR]

Abstract Background Escherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children’s infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran. Methods The E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing. Results Among 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value

Leaf scald caused by Xanthomonas albilineans (Xa) is a major bacterial disease in sugarcane that represents a threat to the global sugar industry. Little is known about the population structure and genetic evolution of this pathogen. In this study, 39 Xa strains were collected from 6 provinces in China. Of these strains, 15 and 24 were isolated from Saccharum spp. hybrid and S. officinarum plants, respectively. Based on multilocus sequence analysis (MLSA), with five housekeeping genes, these strains were clustered into two distinct phylogenetic groups (I and II). Group I included 26 strains from 2 host plants, Saccharum spp. hybrid and S. officinarum collected from 6 provinces, while Group II consisted of 13 strains from S. officinarum plants in the Zhejiang province. Among the 39 Xa strains, nucleotide sequence identities from 5 housekeeping genes were: ABC (99.6–100%), gyrB (99.3–100%), rpoD (98.4–100%), atpD (97.0–100%), and glnA (97.6–100%). These strains were clustered into six groups (A–F), based on the rep-PCR fingerprinting, using primers for ERIC2, BOX A1R, and (GTG)5. UPGMA and PCoA analyses revealed that group A had the most strains (24), followed by group C with 11 strains, while there was 1 strain each in groups B and D–F. Neutral tests showed that the Xa population in S. officinarum had a trend toward population expansion. Selection pressure analysis showed purification selection on five concatenated housekeeping genes from all tested strains. Significant genetic differentiation and infrequent gene flow were found between two Xa populations hosted in Saccharum spp. hybrids and S. officinarum. Altogether, these results provide evidence of obvious genetic divergence and population structures among Xa strains from China. [ABSTRACT FROM AUTHOR]

We compared the virulence profile and REP-PCR genotypes of Escherichia coli strains isolated from subclinical and clinical mastitis cases and dairy farm environments in Minas Gerais State, Brazil, to determine virulence factors and genotypes potentially associated with subclinical persistence in the udder. The virulence profile was obtained by the search for three virulence genes: lpf A (long polar fimbriae), fli C (flagella), and esc N (type III secretion system). Subclinical isolates exhibited mainly the fliC gene (33.33%) and fliC + escN genes (30.30%). Clinical isolates exhibited mainly fliC + escN genes (50%) and environmental isolates the lpfA + escN genes (58.04%). Strains isolated from subclinical mastitis showed 6.75 times more positivity to fli C than environmental isolates. Thirty-four genotypes were observed in the REP-PCR analysis, and clinical mastitis isolates indicated more genetic proximity to dairy farm environment isolates than subclinical mastitis isolates. In conclusion, the results suggested that flagella may be an important virulence factor for mammary persistent E. coli infection in cattle, however, none of the E. coli REP-PCR genotypes were associated with subclinical infection. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is a key nutrient required by plants for their growth and development. A large part of applied phosphatic fertilizer becomes unavailable due to its fixation in soil. In rhizosphere, fluorescent Pseudomonads are instrumental in phosphate solubilization. We isolated Pseudomonads from the rhizosphere of Aloe barbadensis (L.), and screened them for their plant growth promoting ability such as - solubilization of phosphate, auxin production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total of 307 fluorescent Pseudomonas were isolated and screened, of which, 24 isolates showed a strong phosphate solubilizing ability, ranging from 69.71 to 236.73 µg ml−1. The Indole acetic acid (IAA) production by all 24 isolates varied from 28.44 to 151.99 µg ml−1. However, 17 isolates produced ACC-deaminase ranging from 8.19 to 10.27 µmol mg−1 h−1. Analyses of 16S rRNA gene and Pseudomonas-specific genes grouped the isolates in seven different species of fluorescent Pseudomonas. Genotypic analysis based on 16S rDNA-RFLP using restriction enzymes (HaeIII, AluI and MspI) and BOX-PCR generated unique genotype specific pattern. The results suggested that Pseudomonas diversity has no association with microenvironments and the sampling site of the plants. 16S rRNA gene-based diversity indices revealed great diversity among the fluorescent Pseudomonas recovered from the rhizospheric regions of the Aloe. Due to the above plant growth-promoting traits, fluorescent Pseudomonas can be exploited as bio-inoculants for crops, with minimal damage to the environment. [ABSTRACT FROM AUTHOR]

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE. [ABSTRACT FROM AUTHOR]

American foulbrood (AFB) is a serious and near-global disease of honey bees and was recently discovered in South Africa. The causative pathogen, Paenibacillus larvae, comprises various strains. It is currently unknown which of these are present in South Africa and what their distribution is. We address this issue by genotyping P. larvae isolates using repetitive element fingerprinting (rep-PCR) markers and microfabricated chip technology. A total of 71 brood, honey and wax samples were collected opportunistically in South Africa. Twenty-three samples tested positive for P. larvae (32.4%). Diagnostic rep-PCR fingerprinting using ERIC, BOX A1R and MBO REP1 primers found all P. larvae isolates in South Africa to correspond to the ERIC I genotype, with two subtypes revealed, Ab (13 samples) and ab (10 samples). These results are in agreement with the global prevalence of the ERIC I AFB genotype among P. larvae-infected honey bee colonies. [ABSTRACT FROM AUTHOR]

(1) Background: Acinetobacter baumannii is well known as a causative agent of severe hospital-acquired infections, especially in intensive care units. The present study characterised the genetic traits of biofilm-forming carbapenem-resistant A. baumannii (CRAB) clinical isolates. Additionally, this study determined the prevalence of biofilm-producing A. baumannii isolates from a tertiary care hospital and investigated the association of biofilms with the distribution of biofilm-related and antibiotic resistance-associated genotypes. (2) Methods: The 995 non-duplicate A. baumannii isolates were identified, and their susceptibilities to different antibiotics were determined using the disk diffusion method. Using the modified microtiter plate assay, the CRAB isolates were investigated for their biofilm formation ability. Hemolysin and protease activities were determined. CRABs were subjected to polymerase chain reaction (PCR) assays targeting blaVIM, blaNDM, blaIMP, blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, csuE and pgaB genes. Individual CRAB isolates were identified for their DNA fingerprint by repetitive element sequence-based (REP)-PCR. (3) Results: Among all A. baumannii isolates, 172 CRABs were identified. The major antibiotic resistance gene among the CRAB isolates was blaOXA-51-like (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was pgaB (79.65%), followed by csuE (76.74%). Evidence of virulence phenotypes revealed that all CRAB exhibited proteolytic activity; however, only four isolates (2.33%) were positive for the hemolytic-producing phenotype. REP-PCR showed that 172 CRAB isolates can be divided into 36-DNA fingerprint patterns. (4) Conclusions: The predominance of biofilm-producing CRAB isolates identified in this study is concerning. The characterisation of risk factors could aid in controlling the continual selection and spreading of the A. baumannii phenotype in hospitals, thereby improving patient care quality. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas (P.) aeruginosa is one of the most common causes of nosocomial infections. The aim of this study was to explore the antibiotic resistance pattern and genetic association of P. aeruginosa strains isolated from Kermanshah hospitals and their surrounding areas. Methods: The samples included both soils from around three hospitals and clinical specimens of the same hospitals. Once the samples were enriched and the colonies were isolated, the positive samples of this bacterium were confirmed using biochemical tests. Then, antibiotic susceptibility testing (AST) was performed using the disc diffusion method (DDM). Finally, the phylogenetic relationship between the isolates was examined using the rep-PCR technique. In all experiments, the standard strain (ATCC:27853) was used as a positive control. Results: Thirteen and 36 (27 urines, 7 burns and 2 septa) isolates were detected from environmental and clinical samples, respectively. Results showed that clinical strains had the highest resistance to ceftriaxone (94.44%) and the lowest resistance to imipenem (72.2%). On the other hand, environmental strains around the hospital showed the highest resistance to amikacin (92.3%) and the lowest resistance to imipenem (53.84%). Overall, 46 patterns were observed in the strains. The dendrogram indicated that a number of clinical and environmental strains had close genetic similarities. Strains 35C and 9E (clinical strain 35 and environmental strain 9) were 100% similar, which could imply that they originated from a clonal strain. Also, strains 3C, 26C, and 13E were 100% similar. Conclusion: Despite the differences in antibiotic resistance pattern between hospital and environmental isolates, there were genetic similarities between the two isolates. [ABSTRACT FROM AUTHOR]

The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality (Bačka, Vojvodina), known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented (contributing by 19.40% and 17.91%, respectively). Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes (proA, dnaX, icdA, and mdh) and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia. [ABSTRACT FROM AUTHOR]

In this work, the antibiotic resistance, biofilm formation capability, and clonal relatedness of 50 A. baumannii isolates collected from three hospitals in Ardabil city, Iran, were evaluated. Antibiotic sensitivity and biofilm formation of isolates were determined by disk diffusion and microtiter-plate methods, respectively. Molecular typing of isolates was also performed using repetitive sequence-based PCR (REP-PCR). The majority of isolates were resistant to cephems, aminoglycosides, and carbapenems, with 80 % classified as multi-drug resistant (MDR). While, only isolates collected from blood and tracheal were resistant to colistin. Additionally, 42 isolates (84 %) had biofilm formation capability. According to rep-PCR results, 34 isolates showed similar banding patterns, while 16 isolates had unique banding patterns. Finally, based on the molecular analysis, there was a direct relationship between biofilm formation and the antibiotic resistance of isolates. In other words, MDR isolates had a higher ability to form biofilm. • The clonal characteristics of Acinetobacter baumannii isolates were examined. • There was direct relationship between biofilm formation and antibiotic resistance. • Antibiotic colistin can be successfully used against the growth of isolates. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.

Laguna Lake is an economically important resource in the Philippines, with reports of declining water quality due to fecal pollution. Currently, monitoring methods rely on counting fecal indicator bacteria, which does not supply information on potential sources of contamination. In this study, we predicted sources of Escherichia coli in lake stations and tributaries by establishing a fecal source library composed of rep-PCR DNA fingerprints of human, cattle, swine, poultry, and sewage samples (n = 1,408). We also evaluated three statistical methods for predicting fecal contamination sources in surface waters. Random forest (RF) outperformed k-nearest neighbors and discriminant analysis of principal components in terms of average rates of correct classification in two- (84.85%), three- (82.45%), and five-way (74.77%) categorical splits. Overall, RF exhibited the most balanced prediction, which is crucial for disproportionate libraries. Source tracking of environmental isolates (n = 332) revealed the dominance of sewage (47.59%) followed by human sources (29.22%), poultry (12.65%), swine (7.23%), and cattle (3.31%) using RF. This study demonstrates the promising utility of a library-dependent method in augmenting current monitoring systems for source attribution of fecal contamination in Laguna Lake. This is also the first known report of microbial source tracking using rep-PCR conducted in surface waters of the Laguna Lake watershed. HIGHLIGHTS DNA fingerprinting of E. coli, coupled with machine learning algorithms, can be used to discriminate fecal pollution sources in Laguna Lake, Philippines.; The majority of E. coli isolates can be attributed to sewage contamination, followed by human and agricultural sources.; Source-tracking methods can empower local agencies responsible for water quality management to minimize public health and economic risks.;

Japanese quince trees are grown as ornamental plants in Iran, in parks and in orchards close to stone fruit and pome fruit trees. Shoots of Japanese quince (Chaenomeles japonica) showing sunken brown canker symptoms were observed and collected near Sari, the center of Mazandaran province in the North of Iran, during the 2016 growing season. Gram negative bacteria isolated from symptomatic tissues were similar to Pseudomonas syringae pv. syringae (Pss) were pathogenic on Japanese quince and on quince (Cydonia oblonga) seedlings after artificial inoculation, and were re-isolated from diseased hosts. Phylogenetic tree construction using partial sequences of ITS and rpoD genes showed that the Japanese quince isolates were in the same clade as Pss strains. The isolates had ice nucleation activity, and the InaK gene was amplified successfully. According to the results of phenotypic and genotypic characteristics, genomic DNA fingerprinting using REP-PCR, BOX-PCR and IS50-PCR and isolation of total cell proteins, we conclude that Pss is the causal agent of canker of the Japanese quince trees. Therefore, Japanese quince is a new host for Pss causing bacterial canker on many different host plants. [ABSTRACT FROM AUTHOR]

There is increasing evidence showing that microbial dysbiosis impacts the health and cancer risk of the host. An association between adherent-invasive Escherichia coli (AIEC) and colorectal cancer (CRC) has been revealed. Cyclomodulins (CMs) have been receiving increasing attention for carcinogenic changes. In this study, the incidence and features of intracellular AIEC and cyclomodulin-encoding genes were investigated and the phylogenetic grouping and genetic relatedness were evaluated. E. coli strains were isolated from the colorectal biopsies. Adhesion and invasion assays and intramacrophage cell survival test were performed to separate the AIEC isolates. Virulence genotyping for the genes htrA, dsbA, chuA, and lpfA and the cyclomodulin toxins was also conducted. In addition, phylogenetic grouping of the isolates was determined. Subsequently, repetitive element sequence-based PCR (rep-PCR) fingerprinting was performed. A total of 24 AIEC pathovars were isolated from 150 patients. The prevalence rates of htr, dsbA, and lpfA were 70.83% and that of chuA was 91.66%. The frequencies of the cyclomodulin toxins were as follows: cnf1, 29.2%; cnf2, 25%; colibactin, 29.2%; and cdt, 4.2%; cif was not found. Among the AIEC isolates, 4.2%, 4.2%, 54.2%, 29.2%, and 8.3% with phylotypes A or C, B1, B2, D, and E were identified, respectively. Left-sided colon carcinoma and adenocarcinoma T=1 stage (CRC2) were colonized by B2 phylogroup AIEC-producing CMs more often than the samples from the other groups. Close genetic relatedness was observed in AIEC isolates with rep-PCR. [ABSTRACT FROM AUTHOR]

This study aimed to compare the phenotypic and genotypic characteristics, and pathogenicity of Edwardsiella ictaluri derived from several catfish farming in Indonesia. The isolated bacteria were from West Java, East Java, South Sumatra, Jambi, Lampung, Riau and South Kalimantan. The phenotypic analysis based on conventional biochemical characteristics, API 20E and enzyme activity by APIzym showed that the E. ictaluri from various locations in Indonesia had the different growth patterns, ability to hydrolyze sugar (saccharose and amylase), hydrolyzed gelatin, lysed blood agar media and leucine and valine arylamidase enzyme activity. The sensitivity antibiotic testing showed most of the tested isolates were multi‐drug resistance to five antimicrobial classes (macrolides, lincosamides, rifamycin, penicillins and quinolones). The phylogenetic analysis with sequencing 16S rRNA had 99.56%–100% similarity to E. ictaluri. The genotypic analysis of the similarity strains with Genetic Fingerprinting Repetitive sequence‐mediated PCR (Rep‐PCR) denoted different genotype profiles of each primer ERIC 1 ERIC II, BOX and GTG5. The protein profiles were analysed using the SDS‐PAGE method. The whole‐cell isolates of E. ictaluri coded PJbH, P and PBm1G had outer membrane protein (OMP) and riboflavin biosynthesis protein (RibD). The pathogenicity test led to high mortality for 97.78%–100% in the whole‐cell treatment, while the low mortality was 6.67% in the ECP treatment. These results concluded that E. ictaluri isolates from various farming in Indonesia had different phenotypic and genotypic variations and protein components of E. ictaluri cells proved to be virulent and played a role in the pathogenesis of E. ictaluri infection in catfish. [ABSTRACT FROM AUTHOR]

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Japanese quince trees are grown as ornamental plants in Iran, in parks and in orchards close to stone fruit and pome fruit trees. Shoots of Japanese quince (Chaenomeles japonica) showing sunken brown canker symptoms were observed and collected near Sari, the center of Mazandaran province in the North of Iran, during the 2016 growing season. Gram negative bacteria isolated from symptomatic tissues were similar to Pseudomonas syringae pv. syringae (Pss) were pathogenic on Japanese quince and on quince (Cydonia oblonga) seedlings after artificial inoculation, and were re-isolated from diseased hosts. Phylogenetic tree construction using partial sequences of ITS and rpoD genes showed that the Japanese quince isolates were in the same clade as Pss strains. The isolates had ice nucleation activity, and the InaK gene was amplified successfully. According to the results of phenotypic and genotypic characteristics, genomic DNA fingerprinting using REP-PCR, BOX-PCR and IS50-PCR and isolation of total cell proteins, we conclude that Pss is the causal agent of canker of the Japanese quince trees. Therefore, Japanese quince is a new host for Pss causing bacterial canker on many different host plants.

There is increasing evidence showing that microbial dysbiosis impacts the health and cancer risk of the host. An association between adherent–invasive Escherichia coli (AIEC) and colorectal cancer (CRC) has been revealed. Cyclomodulins (CMs) have been receiving increasing attention for carcinogenic changes. In this study, the incidence and features of intracellular AIEC and cyclomodulin-encoding genes were investigated and the phylogenetic grouping and genetic relatedness were evaluated. E. coli strains were isolated from the colorectal biopsies. Adhesion and invasion assays and intramacrophage cell survival test were performed to separate the AIEC isolates. Virulence genotyping for the genes htrA, dsbA, chuA, and lpfA and the cyclomodulin toxins was also conducted. In addition, phylogenetic grouping of the isolates was determined. Subsequently, repetitive element sequence-based PCR (rep-PCR) fingerprinting was performed. A total of 24 AIEC pathovars were isolated from 150 patients. The prevalence rates of htr, dsbA, and lpfA were 70.83% and that of chuA was 91.66%. The frequencies of the cyclomodulin toxins were as follows: cnf1, 29.2%; cnf2, 25%; colibactin, 29.2%; and cdt, 4.2%; cif was not found. Among the AIEC isolates, 4.2%, 4.2%, 54.2%, 29.2%, and 8.3% with phylotypes A or C, B1, B2, D, and E were identified, respectively. Left-sided colon carcinoma and adenocarcinoma T≥1 stage (CRC2) were colonized by B2 phylogroup AIEC-producing CMs more often than the samples from the other groups. Close genetic relatedness was observed in AIEC isolates with rep-PCR.

Bacillus pumilus is the causal agent of trunk bulges disease affecting rubber and rubberwood quality and yield production. In this study, B. pumilus and other closely related species were included in B. pumilus group, as they shared over 99.5% similarity from 16S rRNA analysis. Multilocus sequence analysis (MLSA) of five housekeeping genes and repetitive elements-based polymerase chain reaction (rep-PCR) using REP, ERIC, and BOX primers conducted to analyze the diversity and systematic relationships of 20 isolates of B. pumilus group from four rubber tree plantations in Peninsular Malaysia (Serdang, Tanah Merah, Baling, and Rawang). Multi rep-PCR results revealed the genetic profiling among the B. pumilus group isolates, while MLSA results showed 98-100% similarity across the 20 isolates of B. pumilus group species. These 20 isolates, formerly established as B. pumilus, were found not to be grouped with B. pumilus. However, being distributed within distinctive groups of the B. pumilus group comprising of two clusters, A and B. Cluster A contained of 17 isolates close to B. altitudinis, whereas Cluster B consisted of three isolates attributed to B. safensis. This is the first MLSA and rep-PCR study on B. pumilus group, which provides an in-depth understanding of the diversity of these rubber-pathogenic isolates in Malaysia.

Leaf scald caused by Xanthomonas albilineans (Xa) is a major bacterial disease in sugarcane that represents a threat to the global sugar industry. Little is known about the population structure and genetic evolution of this pathogen. In this study, 39 Xa strains were collected from 6 provinces in China. Of these strains, 15 and 24 were isolated from Saccharum spp. hybrid and S. officinarum plants, respectively. Based on multilocus sequence analysis (MLSA), with five housekeeping genes, these strains were clustered into two distinct phylogenetic groups (I and II). Group I included 26 strains from 2 host plants, Saccharum spp. hybrid and S. officinarum collected from 6 provinces, while Group II consisted of 13 strains from S. officinarum plants in the Zhejiang province. Among the 39 Xa strains, nucleotide sequence identities from 5 housekeeping genes were: ABC (99.6–100%), gyrB (99.3–100%), rpoD (98.4–100%), atpD (97.0–100%), and glnA (97.6–100%). These strains were clustered into six groups (A–F), based on the rep-PCR fingerprinting, using primers for ERIC2, BOX A1R, and (GTG)5. UPGMA and PCoA analyses revealed that group A had the most strains (24), followed by group C with 11 strains, while there was 1 strain each in groups B and D–F. Neutral tests showed that the Xa population in S. officinarum had a trend toward population expansion. Selection pressure analysis showed purification selection on five concatenated housekeeping genes from all tested strains. Significant genetic differentiation and infrequent gene flow were found between two Xa populations hosted in Saccharum spp. hybrids and S. officinarum. Altogether, these results provide evidence of obvious genetic divergence and population structures among Xa strains from China.

This study investigated the distribution and genetic diversity of the indigenous Bradyrhizobium spp. in the Korean agricultural field. A total of 254 Bradyrhizobium strains were isolated from 97 soybean samples (9 cultivars) collected in 14 regions. B. elkanii dominated in the southern regions, while B. diazoefficiens dominated in most central and northern regions. Through non-parametric multidimensional scaling (NMDS) analysis, we confirmed the possibility that environmental factors such as annual average temperature and soybean cultivars might affect the distribution of Bradyrhizobium spp. in some regions. The DNA fingerprint using repetitive DNA sequences showed the genetic diversity among the Bradyrhizobium strains isolated from the different regions. Clustering the strains based on the genetic diversity indicated that Bradyrhizobium spp. grouped into different clusters depending on geographic location. This study suggests that the Korean indigenous Bradyrhizobium spp. distribute differently according to the geographical feature, and the high genetic diversity of each strain attribute to the geographic location. [ABSTRACT FROM AUTHOR]

Fusarium proliferatum known as a pathogen of sheath rot disease. This study aims to determine the genetic diversity by rep-PCR and determine the relationship between the molecular diversity data and the in vitro environmental treatment by using light, pH and temperature and rice variety test. The rep-PCR amplification demonstrated that F. proliferatum was categorized into two groups. Based on the mycelial weight and the growth rate parameters, the 12light/12dark, pH 8, and 25 °C sub-treatments showed the highest outcomes, while in continuous light, pH 6, and 25 °C sub-treatments got the highest spore production. The Inpari 30 rice variety showed the highest disease severity results for F. proliferatum infection. Both the results of the F. proliferatum groups did not affect the fungal growth and fungal infection in several tested rice varieties. These findings suggest that each isolate has a different ability to grow, produce mycelium and spores, and infect a specific rice variety. [ABSTRACT FROM AUTHOR]

Background & aim: Increasing food consumption outdoors in different societies has raised the risk of transmission of foodborne pathogens as a global health problem. Molecular typing methods such as REP-PCR produced DNA profiles for differentiation and characterization of pathogenic strains. The aim of the present study was to evaluate the accuracy of molecular fingerprinting method based on repeated sequences (rep-PCR) to determine the affinities between different of Enterococcus faecium isolated from beef meat as a cause of foodborne infections. Methods: The present cross-sectional descriptive-analytical study was conducted in 2018 on 80 meat samples examined by biochemical and molecular methods for the presence of Enterococcus faecium. The molecular pattern of DNA fragments was determined based on the presence or absence of bands and their size in gel electrophoresis. The collected data were analyzed using NTSYS software version 2.02e and Cofenet correlation coefficient. Results: Of the total 80 samples, 66 (82.5%) were identified as Enterococcus, while 30 (45.45%) were Enterococcus faecium. Based on Genetic Classification by Rep-PCR, 30 isolates of Enterococcus faecium were included in 18 profiles. Placement of the studied isolates in several subgroups showed the acceptable discrimination power of Rep-PCR technique in genotyping of Enterococcus faecium. Conclusion: The results of the present study revealed that Rep-PCR is a simple, fast, and highly dispersive method to describe the genetic diversity of Enterococcus faecium strains.and method with high dispersal ability to characterize the genetic diversity of Enterococcus faecium strains.

Research background. As fermentation is an integral feature of both, dry sausage and cheese production, this has led to the evaluation of bacterial cultures Lactococcus lactis ssp. cremoris (LL8307) and Enterococcus durans (ED0207) originally isolated from artisanal Croatian hard type cheese to diversify the range of flavours of dry fermented sausages and to increase their microbiological safety. Both strains were chosen for their high or medium acidifying, proteolytic and/or lipolytic activity and bioprotective potential after step-by-step selection of wild isolates. Therefore, this study aims to evaluate the survival rate of selected starter cultures in wild boar meat sausages during the ripening period of 40 days at a local small-scale facility under artisanal conditions as well as their influence on sausage quality parameters. Experimental approach. Safety, biotechnological and probiotic properties of twenty-three enterococcal and lactococcal isolates of dairy origin were studied. Based on the results, two best candidates were selected and added to the meat batter during the artisanal wild boar meat sausage preparation where their survival rate, effect on physicochemical, microbiological and sensorial properties and histamine content were evaluated. Results and conclusions. As revealed by repetitive element-polymerase chain reaction (rep-PCR), native starter cultures survived up to 15 days of ripening and were either absent from (LL8307) or reduced by 80% (ED0207) in final products. The application of native starter cultures rapidly decreased pH (p

Background and Objectives: Bacterial stripe caused by Xanthomonas translucens is one of the most important diseases of wheat worldwide. The disease was first reported on barley in America in 1917. In Iran, it was reported for the first time on wheat and barley in Kerman province in 1987. Based on the standards for naming pathovars, the currently accepted pathovars as pathogens infecting cereals include pv. cerealis, pv. translucens, pv. undulosa, and pv. secalis. Following climate changes, especially heavy rainfalls in recent years, and probably the transmission of infected seeds, the disease has shown significant outbreaks and damages on wheat in Kermanshah province, Iran. The present study was performed to (I) characterize the pathogen in Kermanshah province, (II) determine pathogen distribution, and (III) assess the genetic diversity of the strains. Materials and Methods: Wheat seeds and leaves showing symptoms of the disease were collected from major wheat-growing areas in Kermanshah province during 2019 and 2020. In the laboratory, samples were rinsed with tap water and were macerated into a few drops of sterile distilled water. Two loops full of the resulting suspension were streaked on the nutrient agar medium. Plates were incubated at 25°C and pale yellowish colonies were purified. Physiological and biochemical tests were performed to identify and characterize 140 isolates. To determine the pathovar(s) of the pathogen, the pathogenicity of the strains was tested on wheat, barley, rye, and triticale in the greenhouse. Nearly complete 16S rRNA sequences were determined for two strains representatives of two pathovars distinguished by pathogenicity test using 27F and 1492R primers. Genetic diversity among the isolates was evaluated in rep-PCR utilizing BOX and ERIC primers. Results: Based on physiological and biochemical tests, Xanthomonas strains from wheat in Kermanshah province were identified as X. translucens. Pathogenicity tests revealed two groups among the strains. Members of the first group induced disease symptoms on wheat and triticale exclusively and were identified as pv. undulosa, while the strains belonging to the second group infected wheat, barley, rye, and triticale, and were identified as pv. cerealis. The rep-PCR using ERIC and BOX primers demonstrated genetic variation among the strains. However, there was no significant difference between the results obtained by ERIC- and BOX-PCR. Nearly complete 16S rRNA sequences were determined as the representative strains of two pathovars distinguished by pathogenicity test and based on the comparison of sequences with the GenBank database. The two strains matched X. translucens with 100% similarity. The nucleotide sequences of the pathogen strains were deposited in the GenBank database under the accession numbers MW173461 and MW193070. Discussion: According to the findings of this study, X. translucens strains collected from wheat in Kermanshah province belonged to pathovars undulosa and cerealis. Considering the wide host range of these pathovars, the disease could be found on other cereals and grasses belonging to the Poaceae in the province and this subject must be taken into consideration in future studies. Although rep-PCR showed significant diversity among the strains, there was no correlation between the rep-PCR groups and the pathovars identified in the pathogenicity test or the geographic origin of the strains. Biochemical and physiological tests, as well as rep-PCR and 16S rRNA sequencing, could not distinguish the two pathovars. This confirms that pathogenicity tests and host range are still the main methods for determining pathogen pathovar(s). [ABSTRACT FROM AUTHOR]

Background: Serratia marcescens (S. marcescens) is associated with nosocomial infections with significant morbidity and mortality in the neonatal intensive care units (NICU). We describe the control of a multi-clonal S. marcescens infections outbreak in our tertiary-level NICU and the application of molecular typing using repetitive element palindromic PCR (rep-PCR) and next generation sequencing (NGS) in the investigation. Methods: Outbreak investigation was performed where clinical, spatial and epidemiologic links were established. Screening of all infants in the NICU and the environment was performed. Rep-PCR and NGS methods were used to identify potential environmental sources of infections and clustering among cases. Results: Eleven cases were detected during the outbreak period: mean gestational age 27 weeks (range: 24–32), predominantly male (82%), mean age of infection 24 days (range: 6–51). Six infants were treated for conjunctivitis and one for bacteraemia. Identification of colonized infant via a point prevalence survey and cohorting of all infected/colonized patients were implemented. We performed environmental swabbing of surfaces, water outlets, chlorhexidine hand wash solutions and hand hygiene hand rubs. Both rep-PCR and NGS classified the 11 case isolates into 5 types. No point source was identified except for a single positive environmental isolate from a sink which was clonally distinct from the cases. Conclusion: Identification and cohorting of infected/colonized patient was important in the control of S. marcescens outbreak in the NICU. The utility of rep-PCR was comparable to NGS in providing molecular information to develop S. marcescens outbreak control strategies.

In this study, the susceptibility to cephalosporins of 74 Salmonella isolates, including 16 isolates from farm animals and 58 isolates from poultry was assessed by the disc diffusion method. ESBL production was evaluated by combined disc diffusion method (CDDM) and double disc synergy test (DDST). The genetic relatedness of isolates was investigated by the Rep-PCR method. The highest prevalence of resistance was observed against cefotaxime (27%) and the least – against cefixime (4%). None of the isolates was ESBL positive. The Rep-PCR generated 54 reproducible fingerprint patterns for all isolates and grouped them in four clusters and six singletons. Due to public health risk of cross contamination, it is important to have sufficient information on the occurrence of these resis-tant isolates.