High prevalence of plasmid-mediated quinolone resistance in escherichia coli strains producing extended-spectrum beta-lactamases isolated from faeces and urine of pregnant women with acute cystitis.

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 ß-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.

A comparative study of genotyping and antimicrobial resistance between carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii isolates at a tertiary pediatric hospital in China.

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. Results: The rates of CRKP and CRAB ranged from 15.8% to 37.0% at the hospital. Forty-nine and sixteen REP genotypes were identified among the 96 and 48 CRKP and CRAB isolates tested, respectively. The CRKP isolates showed more genetic diversity than the CRAB isolates. Of the 96 CRKP isolates, 69 (72%) produced Class B carbapenemases. However, all 48 CRAB isolates produced Class D carbapenemase or extended-spectrum ß-lactamases (ESBL) combined with the downregulation of membrane pore proteins. Furthermore, the carbapenemase genes bla KPC, bla IMP, and bla NDM were detected in CRKP isolates. However, CRAB isolates were all positive for the bla VIM, bla OXA-23, and bla OXA-51 genes. 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.

Evaluation of molecular typing methods for some scab-causing Streptomyces strains from Turkey.

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.

A polyphasic molecular approach to characterize a collection of grapevine endophytic bacteria with bioprotective potential.

AIMS: The work presented here was conducted to characterize the biodiversity of a collection of bacterial isolates, mainly wood endophytes, as part of a research project focused on exploring their bioprotective potential for postharvest biological control of fruits. METHODS AND RESULTS: This work was the basis for the development of a tailored method combining 16S rDNA sequencing and Rep-PCR to differentiate the isolates and identify them to genus level or below. More than one hundred isolates obtained from wood and roots of different grapevine genotypes were cultured on appropriate growth media and then subjected to the specified multistep molecular identification. CONCLUSIONS: We have obtained good dereplication for grapevine-endophytic bacteria, together with reliable genetic identification. Both are essential prerequisites to properly characterize a biome bank and, at the same time, beneficial prerequisites to subsequently perform a correct bioprotection assessment.

Genetic diversity, mating type and pathogenicity of two Phytophthora species infecting black pepper in India.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03843-1.

Genetic Divergence and Population Structure of Xanthomonas albilineans Strains Infecting Saccharum spp. Hybrid and Saccharum officinarum.

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.

Molecular epidemiology typing of blaOXA-48 and blaNDM-1 producing Klebsiella pneumoniae causing nosocomial infection.

In this study the antibiotic susceptibility pattern and bla genes were characterized in Klebsiella pneumoniae clinical isolates that fingerprinted by rep-PCR and PFGE methods at Kurdistan Province, Iran. A total of 70 K. pneumoniae were isolated from clinical samples to detect the antimicrobial susceptibility, carbapenemase and MBL-producing isolates. The PCR assay was used to identify the bla genes. Isolates were typed by PFGE and Rep-PCR methods. The highest and lowest rates of resistance were observed in cefotaxime (67.1%) and imipenem (8.6%), respectively. The rate of blaNDM-1 and blaOXA-48 genes were 1 (1.4%) and 14 (20%) isolates, respectively. All were classified in 27 clusters by rep-PCR and 39 PFGE types. The low frequency of carbapenemase and MBL genes in this study are epidemiologically important.

Levilactobacillus brevis, autochthonous to cucumber fermentation, is unable to utilize citric acid and encodes for a putative 1,2-propanediol utilization microcompartment.

The metabolic versatility of Levilactobacillus brevis, a heterofermentative lactic acid bacterium, could benefit environmentally compatible and low salt cucumber fermentation. The biodiversity of Lvb. brevis autochthonous to cucumber fermentation was studied using genotypic and phenotypic analyses to identify unique adjunct cultures. A group of 131 isolates autochthonous to industrial fermentations was screened using rep-PCR-(GTG)5 and a fermentation ability assay under varied combinations of salt (0 or 6%), initial pH (4.0 or 5.2), and temperature (15 or 30°C). No apparent similarities were observed among the seven and nine clusters in the genotypic and phenotypic dendrograms, respectively. A total of 14 isolates representing the observed biodiversity were subjected to comparative genome analysis. The autochthonous Lvb. brevis clustered apart from allochthonous isolates, as their genomes lack templates for citrate lyase, several putative hypothetical proteins, and some plasmid- and phage-associated proteins. Four and two representative autochthonous and allochthonous Lvb. brevis, respectively, were subjected to phenotype microarray analysis using an Omnilog. Growth of all Lvb. brevis strains was supported to various levels by glucose, fructose, gentiobiose, 1,2-propanediol, and propionic acid, whereas the allochthonous isolate ATCC14890 was unique in utilizing citric acid. All the Lvb. brevis genomes encode for 1,2-propanediol utilization microcompartments. This study identified a unique Lvb. brevis strain, autochthonous to cucumber, as a potential functional adjunct culture for commercial fermentation that is distinct in metabolic activities from allochthonous isolates of the same species.

Molecular detection, typing, and virulence potential of Salmonella Serotypes isolated from poultry feeds.

Salmonella contamination in poultry feed is one of the main issues in poultry industry and public health. The aim of the present study was molecular detection and typing of Salmonella serotypes isolated from poultry feeds. Moreover, we determined the antibiotic resistance pattern and the ability of biofilm formation in the serotypes. To this end, eighty feed samples were collected from aviculture depots. Salmonella serotypes were identified by culture and PCR methods. For serological identification, a slide agglutination test was used. BOXAIR and rep-PCR methods were applied to evaluate the diversity of serotypes. The disc diffusion method was performed to evaluate the antibiotic susceptibility of serotypes to sixteen antibiotics. Biofilm formation was also assessed by the microtiter-plate test. From a total of 80 feed samples, 30 samples were contaminated with Salmonella spp., which were divided into 5 different serotypes belonging to B, C, and D serogroups. BOXAIR-PCR (D value [DI] 0.985) and rep-PCR (DI 0.991) fingerprinting of isolates revealed 23 and 19 reproducible fingerprint patterns, respectively. A higher antibiotic resistance was observed to ampicillin and doxycycline (100% each), followed by chloramphenicol (83.33%) and tetracycline (73.33%). Multidrug resistance (MDR) was detected in all Salmonella serotypes. Half of the serotypes possessed the ability of biofilm formation with varied adhesion strengths. These results revealed the high and unexpected prevalence of Salmonella serotypes in poultry feed with MDR and biofilm formation ability. BOXAIR and rep-PCR revealed a high diversity of Salmonella serotypes in feeds and subsequently indicated variation in the source of Salmonella spp. The unknown sources harboring high diversity of Salmonella serotypes indicated poor control, which could cause problems for feed manufacturing.

Flagella are an important virulence factor in the subclinical persistence of Escherichia coli in bovine mammary gland.

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: lpfA (long polar fimbriae), fliC (flagella), and escN (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 fliC 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.

Phenotypic and genetic screening of Klebsiella pneumoniae isolates from human UTI patients for beta-lactamases and their genetic diversity analysis by ERIC and REP PCRs.

Klebsiella pneumoniae is one of the major nosocomial pathogens responsible for pneumoniae, septicaemia, liver abscesses, and urinary tract infections. Coordinated efforts by antibiotic stewardship and clinicians are underway to curtail the emergence of antibiotic-resistant strains. The objective of the present study is to characterize K. pneumoniae strains through antibiotic resistance screening for production of beta-lactamases (ß-lactamases) such as extended spectrum beta lactamases (ESBLs), AmpC ß-lactamases, and carbapenemases by phenotypic and genotypic methods and genetic fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and repetitive element palindromic PCR (REP-PCR). A total of 85 K. pneumoniae strains isolated from 504 human urinary tract infections (UTI) were used in this study. Only 76 isolates showed positive in phenotypic screening test (PST), while combination disc method (CDM) as phenotypic confirmatory test (PCT) confirmed 72 isolates as ESBL producers. One or more ß-lactamase genes were detected by PCR in 66 isolates (91.66%, 66/72) with blaTEM gene being the most predominant (75.75%, 50/66). AmpC genes could be detected in 21 isolates (31.8%, 21/66) with FOX gene being the predominant (24.24%, 16/66), whereas NDM-I was detected in a single strain (1.51%, 1/66). Genetic fingerprinting using ERIC-PCR and REP-PCR revealed wide heterogeneity among ß-lactamase producing isolates with discriminatory power of 0.9995 and 1, respectively.

A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting.

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95-98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare.

Exploring yeast diversity of dry-salted naturally black olives from Greek retail outlets with culture dependent and independent molecular methods.

In the present study, the physicochemical (pH, water activity, moisture content, salt concentration) classical plate counts (total viable counts, yeasts, lactic acid bacteria, Staphylococcus aureus, Pseudomonas spp., Enterobacteriaceae) and amplicon sequencing of naturally black dry-salted olives obtained from different retail outlets of the Greek market were investigated. According to the results, the values of the physicochemical characteristics presented great variability among the samples. Specifically, pH and water activity (aw) values ranged between 4.0 and 5.0, as well as between 0.58 and 0.91, respectively. Moisture content varied between 17.3 and 56.7 % (g Η2Ο/100 g of olive pulp), whereas salt concentration ranged from 5.26 to 9.15 % (g NaCl/100 g of olive pulp). No lactic acid bacteria, S. aureus, Pseudomonas spp. and Enterobacteriaceae were detected. The mycobiota consisted of yeasts that were further characterized and identified by culture-dependent (rep-PCR, ITS-PCR, and RFLP) and amplicon target sequencing (ATS). Pichia membranifaciens, Candida sorbosivorans, Citeromyces nyonsensis, Candida etchelsii, Wickerhamomyces subpelliculosus, Candida apicola, Wickerhamomyces anomalus, Torulaspora delbrueckii and Candida versatilis were the dominant species according to ITS sequencing (culture-dependent), while ATS revealed the dominance of C. etchelsii, Pichia triangularis, P. membranifaciens, and C. versatilis among samples. The results of this study demonstrated considerable variability in quality attributes among the different commercial samples of dry-salted olives, reflecting a lack of standardization in the processing of this commercial style. However, the majority of the samples were characterized by satisfactory microbiological and hygienic quality and complied with the requirements of the trade standard for table olives of the International Olive Council (IOC) for this processing style in terms of salt concentration. In addition, the diversity of yeast species was elucidated for the first time in commercially available products, increasing our knowledge on the microbial ecology of this traditional food. Further investigation into the technological and multifunctional traits of the dominant yeast species may result in better control during dry-salting and enhance the quality and shelf-life of the final product.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

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.

Phenotypic and Genotypic Investigation of Carbapenem-Resistant Acinetobacter baumannii in Maharaj Nakhon Si Thammarat Hospital, Thailand.

(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.

Diversity of Bacterial Soft Rot-Causing Pectobacterium Species Affecting Cabbage in Serbia.

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 (Backa, 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.

The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131.

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 < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern. CONCLUSIONS: Our results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

High Prevalence of Panton-valentine Leukocidin-encoding Genes in Methicillin-resistant Staphylococcus aureus Isolated from Inpatients with Invasive Infections at a University Hospital in Southern Brazil.

BACKGROUND: Staphylococcus aureus is a major cause of a wide diversity of infections in humans, and the expression of Panton-Valentine Leukocidin (PVL) has been associated with severe clinical syndromes. OBJECTIVES: The present study aimed to investigate the prevalence of PVL-encoding genes in S. aureus isolated from clinical samples of inpatients with invasive infections in a teaching hospital in Southern Brazil. Furthermore, phenotypic and genotypic characteristics of bacterial isolates were analyzed. METHODS: A total of 98 S. aureus isolates recovered from different body sites were characterized according to their antimicrobial susceptibility profile, methicillin-resistance and SCCmec typing, genetic relatedness and occurrence of virulence-encoding genes, such as icaA, lukS-PV/lukF-PV, and tst. RESULTS: Sixty-eight (69.4%) isolates were classified as methicillin-resistant, and among them, four (5.9%) did not harbor the mecA gene. The mecA-harboring methicillin-resistant S. aureus (MRSA) isolates were grouped into SCCmec types I (6.3%), II (64.1%), III (6.3%), IV (15.6%), V (4.7%), and VI (1.6%). One isolate (1.6%) was classified as non-typeable (NT). Seventy isolates (71.4%) were classified as multidrug-resistant. The overall prevalence of virulence-encoding genes was as follows: icaA, 99.0%; tst, 27.5%; and lukS-PV/lukF-PV, 50.0%. The presence of tst gene was significantly higher (p < 0.001) in methicillin-susceptible S. aureus (MSSA) compared to MRSA isolates. CONCLUSION: The present study reports a high prevalence of multidrug-resistant S. aureus harboring lukS-PV/lukF-PV and tst genes in invasive infections. The continuous monitoring of the antimicrobial susceptibility profile and virulence of S. aureus is an important measure for the control of infections caused by this bacterium.

Phenotypicand Genotypic Characterization of Clinical Isolates of Intracellular Adherent-Invasive Escherichia coli Among Different Stages, Family History, and Treated Colorectal Cancer Patients in Iran.

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.

Innovative Detection of the Quarantine Plant Pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens, Causal Agent of Bacterial Wilt of Leguminous Plants.

Specific, sensitive, and rapid detection of quarantine and regulated plant pathogens is pivotal for the control of the diseases they cause. Here, we describe the PCR-based methods which have been developed for Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), quarantine plant pathogenic bacterium for EU and causal agent of bacterial wilt of Leguminous plants. These methods include an end-point and a real-time PCR test, and a LAMP assay. Their threshold analytical limits range from 100 to 10 fg of DNA template per reaction and are currently used worldwide for routine testing for Cff from laboratory to field scale.