Molecular bacteriology

U rinary tract infections (UTIs) are one of the most prevalent infectious diseases in both hospital and community settings and can lead to a high rate of morbidity and mortality. 1,2 various pathogens are responsible for causing UTIs, of which Escherichia coli account for the majority of infections. 3 among the array of antibiotics commonly used for the treatment of UTIs, β-lactams are the most extensively used agents. 1 However, the emergence of multiple-drug resistant (mdR) strains, particularly extended-spectrum beta-lactamases (eSBls) producing strains, has become a global healthcare concern. 4-8 Recently, the trend of eSBl-producing E. coli strains has increased worldwide, including in Iran. 9,10 eSBls are enzymes capable of hydrolyzing penicillins, broad-spectrum cephalosporins, and monobactams. 11 The specific eSBl-producing strains have different genetic characteristics, which may cause specific pathogenesis characteristics. 4,12 The antibiotic resistance determinants can normally transfer among bacterial strains by different horizontal gene transfer mechanisms, including integrons. 13 These elements are responsible for the integration and dissemination of resistance genes among the bacteria. To date, several classes of integrons have been described based on the amino acid sequences of their integrase genes (intI). 13 Of them, class 1 and 2 integrons are the most prevalent classes of mdR gram-negative bacteria associated with antibiotic treatment failure. 14

Urinary tract infection (UTI) is the second common infection in human which affects millions of people each year (1). Increasing rates of resistance among bacterial uropathogens has caused growing concern in both developed and developing countries. Escherichia coli, the most common member of the family Enterobacteriaceae, accounts for 75-90% of all UTIs in both in-and out-patient settings (2). Understanding molecular mechanisms, through which resistance genes transfer, may contribute to developing new antimicrobial agents as well as preventive measures to stop further dissemination of resistance determinants among pathogens (3).

Peach (Prunus persica L.) fruit rich in mineral, fiber and vitamin source is a major fruit in Khyber Pakhtunkhwa (KPK) and northern areas of Pakistan. With an average production of 30 thousand tons, it is cultivated on an area of 56 thousand hectares in KPK. During a survey of 29 peach orchards in 2014-15 and 2015-16, leaves and fruits with water soaked brown necrotic lesions and stems with canker and gum execution were observed. Mean disease incidence was recorded as 49% on cv. Locally known as 6 ½. A total of 260 samples were collected in December-January (blooming stage) to May (fruiting stage). After isolation on nutrient agar medium formed bacterial colonies were streaked on King's B medium to detect florescence under UV light. Cycloheximide 50mg/l was added to the medium to prevent fungal growth. A total of 32 recovered isolates were gram-negative, levan and tobacco hypersensitivity positive while oxidase, potato soft rot and arginine dihydrolase negative, and confirmed as Pseudomonas syringae. During GAATa scheme; gelatine and aesculin hydrolysis negative while tyrosinase and tartrate positive confirmed 11 isolates were pathovar morsprunorum race 1 . Further identification of P. syringae pv. morsprunorum race 1 was confirmed by PCR amplification of 16S rRNA and gyrB gene . Obtained sequences of 16S rRNA and gyrB gene (Genbank Accession No. KY364875 and MF401398 respectively) showed 96-99% genetic similarity with already reported P. syringae pv. morsprunorum race 1 sequences (Genbank Accession No. KF019104 and HG000214 respectively). Pathogenicity was done by inoculating fresh peach fruits with bacterial suspension (10 8 cfu/ml) of these two strains together with a pathotype strain incubated at 27±2ºC. A set of healthy fruits inoculated with double distilled water was used as a control . 5-7 days after inoculation, water-soaked, brown lesions with yellow margins were observed on the fruits inoculated with bacterial strains. The pathogen was re-isolated and confirmed P. syringae pv. morsprunorum race 1 using LOPAT and GATTa tests. To our knowledge, this is the first report of P. syringae pv. morsprunorum race 1 from KPK province of Pakistan and the information will help in devising quarantine measures to limit the pathogen prevalence.

Peach (Prunus persica L.) fruit rich in mineral, fiber and vitamin source is a major fruit in Khyber Pakhtunkhwa (KPK) and northern areas of Pakistan. With an average production of 30 thousand tons, it is cultivated on an area of 56 thousand hectares in KPK. During a survey of 29 peach orchards in 2014-15 and 2015-16, leaves and fruits with water soaked brown necrotic lesions and stems with canker and gum execution were observed. Mean disease incidence was recorded as 49% on cv. Locally known as 6 ½. A total of 260 samples were collected in December-January (blooming stage) to May (fruiting stage). After isolation on nutrient agar medium formed bacterial colonies were streaked on King's B medium to detect florescence under UV light. Cycloheximide 50mg/l was added to the medium to prevent fungal growth. A total of 32 recovered isolates were gram-negative, levan and tobacco hypersensitivity positive while oxidase, potato soft rot and arginine dihydrolase negative, and confirmed as Pseudomonas syringae. During GAATa scheme; gelatine and aesculin hydrolysis negative while tyrosinase and tartrate positive confirmed 11 isolates were pathovar morsprunorum race 1 . Further identification of P. syringae pv. morsprunorum race 1 was confirmed by PCR amplification of 16S rRNA and gyrB gene . Obtained sequences of 16S rRNA and gyrB gene (Genbank Accession No. KY364875 and MF401398 respectively) showed 96-99% genetic similarity with already reported P. syringae pv. morsprunorum race 1 sequences (Genbank Accession No. KF019104 and HG000214 respectively). Pathogenicity was done by inoculating fresh peach fruits with bacterial suspension (10 8 cfu/ml) of these two strains together with a pathotype strain incubated at 27±2ºC. A set of healthy fruits inoculated with double distilled water was used as a control . 5-7 days after inoculation, water-soaked, brown lesions with yellow margins were observed on the fruits inoculated with bacterial strains. The pathogen was re-isolated and confirmed P. syringae pv. morsprunorum race 1 using LOPAT and GATTa tests. To our knowledge, this is the first report of P. syringae pv. morsprunorum race 1 from KPK province of Pakistan and the information will help in devising quarantine measures to limit the pathogen prevalence.

Contexte : En Ontario, l'infection à Neisseria meningitidis de sérogroupe W (MenW) représente un faible pourcentage de toutes les méningococcies invasives (MI) et entre 2010 et 2014, seulement zéro à trois cas confirmés ont été recensés chaque année. Cependant, entre le mois d'août 2015 et le mois de juin 2016, six cas confirmés par culture de MI de sérogroupe W (MenW) ont été signalés en Ontario. Objectif : Tous les cas de MI de sérogroupe W (MenW) survenus en Ontario entre le 1 er janvier 2009 et le 30 juin 2016 ont été examinés et les souches concernées de N. meningitidis ont été caractérisées. Méthodologie : Les cas de MenW ont été identifiés dans le Système d'information sur la santé publique intégré par Santé publique Ontario. Les isolats de MenW ont été caractérisés au Laboratoire national de microbiologie. Résultats : Sur les treize cas de MI de sérogroupe W (MenW), six étaient dus à des isolats identifiés par typage comme séquence type (ST)-22 complexe clonal (cc), six à des isolats de complexe clonal ST-11, et un à l'isolat de complexe clonal ST-167. La plupart (83 %) des cas de MenW dus au complexe clonal ST-22 sont survenus avant 2012, tandis que les six cas de MenW dus au complexe clonal ST-11 se sont produits depuis mai 2014. Les six isolats de MenW ST-11 se sont avérés clonaux. Conclusion : Il semble qu'une modification génétique des isolats de MenW se soit produite en Ontario en 2014, le clone ST-11 remplaçant le clone ST-22 traditionnel.

Campylobacter enteritis is the leading cause of gastroenteritis in humans worldwide including Bangladesh. The objectives of this study were to estimate the prevalence and antimicrobial-resistance status of Campylobacter spp. in human diarrheal samples collected from Surya Kanta Hospital, Mymensingh, Bangladesh. In this study, we evaluated a total of 330 clinical samples for the presence Campylobacter spp. via cultural and biochemical tests and molecular assays. Furthermore, antimicrobial susceptibility testing for Campylobacter species was accomplished by the standard agar disc diffusion technique against eight commercially available antimicrobial agents. A pretested semistructured questionnaire was used to capture the data on socioanthropological factors from the diarrheal patients. Pearson’s chi-square test was performed, and a p value of <0.05 was considered for the level of significance. Nearly one in three diarrheal patients admitted in this hospital were infected with Campy...

Ralstonia solanacearum , the causative agent of bacterial wilt, is a devastating bacterial plant pathogen with a wide range of hosts. We report here the first draft genome sequences for three strains of Ralstonia solanacearum isolated from infected potato, tomato, and pepper plants in Georgia.

Background Neisseria meningitidis is the leading responsible bacterium of Purpura Fulminans (PF) accounting for two thirds of PF. Skin biopsy is a simple and minimally invasive exam allowing to perform skin culture and polymerase chain reaction (PCR) to detect Neisseria meningitidis. We aimed to assess the sensitivity of skin biopsy in adult patients with meningococcal PF. Methods A 17-year multicenter retrospective cohort study including adult patients admitted to the ICU for a meningococcal PF in whom a skin biopsy with conventional and/or meningococcal PCR was performed. Results Among 306 patients admitted for PF, 195 had a meningococcal PF (64%) with a skin biopsy being performed in 68 (35%) of them. Skin biopsy was performed in median 1 day after the initiation of antibiotic therapy. Standard culture of skin biopsy was performed in 61/68 (90%) patients and grew Neisseria meningitidis in 28 (46%) of them. Neisseria meningitidis PCR on skin biopsy was performed in 51/68 (75%) patients and was positive in 50 (98%) of them. Among these 50 positive meningococcal PCR, five were performed 3 days or more after initiation of antibiotic therapy. Finally, skin biopsy was considered as contributive in 60/68 (88%) patients. Identification of the meningococcal serogroup was obtained with skin biopsy in 48/68 (71%) patients. Conclusions Skin biopsy with conventional culture and meningococcal PCR has a global sensitivity of 88% and should be systematically considered in case of suspected meningococcal PF even after the initiation of antimicrobial treatment.

A hybrid of six (6) 4-hydroxyindole azo compounds was synthesised by the diazotization and coupling strategy via electrophilic aromatic substitution reaction. Characterisation by Infrared and UV-Vis spectroscopic studies was carried out and the antimicrobial activity and structure-activity relationship were explored. Amongst the compounds, 4d was the most active against Pseudomonas aeruginosa than the other organisms from the high-throughput spot culture growth inhibition (HTSPOTi) antimicrobial assay. According to the resistant modulation study, the compounds did not show significant activity against the panel of pathogens used. Furthermore, compounds 4a and 4f inhibited biofilm formation in Pseudomonas aeruginosa and Staphylococcus aureus at 70% (31.25 µg/mL) and 57% (125 (µg/mL) respectively. Additionally, 4c and 4e have biofilm inhibition potential against Pseudomonas aeruginosa and Staphylococcus aureus which are implicated in antimicrobial resistance. Hence, the compounds are ...

Background Myocardial failure has a central role in the complex pathophysiology of septic shock and contributes to organ failure and death. During the sepsis-induced inflammatory process, specific factors are released that depress myocardial contractile function. We aimed to identify these mediators of myocardial depression in meningococcal septic shock.

Dysgonic fermenter 3 (DF-3)-associated bacteremia occurred in a febrile patient with acute myelocytic leukemia during aplasia. Another DF-3 isolate, identical by ribotyping, was grown 10 weeks later from stool collected in the absence of diarrhea. This is the first case in which DF-3 was isolated from blood and stool specimens from the same patient.

The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, efflux pump and biofilm inhibitory activities of bulb extracts of selected Ghanaian shallots Allium cepa var aggregatum were evaluated using the HT-SPOTi assay and other whole-cell phenotypic screening techniques to determine their possible mechanisms of action. Ethanol and aqueous extracts of white A. cepa inhibited the growth of Mycobacterium smegmatis mc2 155 and Escherichia coli, respectively. The majority of the Allium extracts significantly (p < 0.05) exhibited efflux pump inhibitory activity against all the acid-fast, Gram-positive and Gram-negative strains used. Hexane and chloroform extract of the pink A. cepa and the aqueous extract of the white A. cepa significantly inhibited M. smegmatis biofilm formation. For Pseudomonas aeruginosa, the inhibition was observed at 250 µg/mL for the aqueous extract (~77.34%) and 125 µg/mL for the hexane extract (~76.51%). The results suggest...

BackgroundAntimicrobial resistance is a global health challenge. The involvement of bacterial biofilms and efflux pumps in the development of multidrug resistance (MDR) is well established. Medicinal plants have been proposed as alternatives for combating MDR focusing on their bioactive constituents with resistance modulatory activities. This study was aimed at investigating the stem bark ofAidia genipiflorafor bioactive constituents with anti-biofilm, efflux pump inhibition and resistance modulatory activities.MethodThe crude methanol extract was purified by column chromatography and isolated compounds characterized by mass and nuclear magnetic resonance spectrometry. Antibacterial activity was determined by the High-throughput spot culture growth inhibition and the broth micro-dilution assay. The ethidium bromide accumulation assay was used to determine efflux pump inhibition property. Biofilm inhibition was determined in a microplate crystal violet retention assay.ResultsPurifica...

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube À1 by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.

Guillain-Barré syndrome to meningococcal conjugate vaccine: Menveo ® Mots clés Syndrome de Guillain-Barré ; Vaccin méningococcique Keywords Guillain-Barré syndrome; Meningococcal vaccine Abréviations CRP protéine C réactive ECG électrocardiogramme ENMG électroneuromyogramme IIM infection invasive à méningocoque SGB syndrome de Guillain-Barré VAERS vaccine adverse event reporting system * Auteur correspondant.

Objective: To assess the discriminatory power of two genotypic and two phenotypic techniques by analysis of Pseudomonas aeruginosa sputum isolates obtained with long term intervals from 29 independent cystic fibrosis (CF) patients. Methods: Fifty-eight strains of P aeruginosa were subjected t o serotyping and pyocin production was assessed. Arbitrarily primed polymerase chain reaction (AP PCR) and pulsed-field gel electrophoresis (PFGE) were applied in order to detect genetic polymorphisms. Results: From the results of different typing techniques, it appeared that the numbers of separate types varied between 11 and 43, and the percentage of identical P aeruginosa pairs from individual patients varied between 51% and 72%, depending on the test system used. AP PCR and PFGE displayed enhanced resolution when compared to serotyping and pyocin typing; both DNA typing techniques generated concordant results, although differences in resolution are apparent. This results in 15% discordance, which may be the result of differences in the definitions of (sub)clonal relatedness as applied for AP PCR and PFGE, respectively. Conclusions: Molecular typing techniques are superior t o phenotyping where P aeruginosa is concerned. AP PCR is a fast and useful technique for determining clonality among P aeruginosa strains from chronically colonized CF patients. It is clear, however, that the interpretation of data and comparative analysis of PFGE and AP PCR results necessitates additional (international) standardization and the development of practical guidelines.

The aims of this study were to investigate the antibiotics susceptibility, multidrug-resistant (MDR) frequency and the association of integrons with MDR among phylogenic groups of uropathogenic E. coli (UPEC). In total, 176 non-duplicated UPEC isolates were collected from urinary tract infections (UTIs) specimens. The disk diffusion method was performed for determination of antibiotic susceptibility patterns. Phylogenetic grouping and the presence of integron-associated genes (int) were detected by the PCR technique. A high frequency of resistance was observed to cotrimoxazole (96.9%), ampicillin (85%), trimethoprim (80.1%) and cefazolin (79.6%); and 140 isolates (79.5%) were MDR. Carbapenems and fosfomycin were the most effective antibiotics. The majority of isolates (60.8%) belonged to the phylogenic group B2. Integrons were detected in 135 (76.7%) of isolates and, class I was the most common (63.6%) class. MDR isolates were found to be significantly associated with class І integrons. These isolates were found to be closely associated with the phylogenic group D (82%), however, the presence of class І integrons was higher among MDR isolates of the phylogroup B1. This pattern is believed to be due to other mechanisms such as the overexpression of the efflux pumps. Our findings show a significant correlation between MDR and the presence of class І integron. We conclude that class 1 integron plays an important role in the development of MDR UPEC, especially among the phylogroup B1.

As part of Federal Government policy on Small Medium Enterprises in Nigeria, a lot of small scale businesses have sprung up including soap making industries using indigenous contents. The ability of indigenously manufactured soaps to remove germs and dirt is paramount. An in vitro evaluation of antibacterial activity of twelve randomly collected indigenously made soaps in Gombe metropolis, Nigeria was conducted using agar well diffusion method against strains of reference microbes viz; Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Klebsiella pneumonia being human skin bacteria, followed by time kill kinetic assay to determine the pharmaco-dynamics of active soaps against susceptible test organisms. The results obtained show that six of the soaps exhibited antibacterial activity with varying degree of zones of inhibition. S. aureus was the most susceptible amongst the organisms while E. coli and P. aeruginosa were the least susceptible microbes. The time kill kinetic assay shows that the bactericidal effect of the soaps is dose and contact time dependent as the susceptible organisms were eliminated after 8 h exposure. The antibacterial activities exhibited by these soaps suggest them as potential candidate in bio-prospecting for antibacterial.

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacterpositive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases.

Selective activation …o fb iosynthetic gene clusters in Dhfq strains of different proteobacteria facilitates isolation and bioactivity testing of (exclusively produced) natural products as described by H. B. Bode et al. in their Research Article on page 18957. Easy Promoter Activated Compound Identification (easyPACId) activates even silent gene clusters,s ymbolized by the closed Advent calendar packages,r evealing the identity as well as bioactivity of the corresponding natural products directly from the production culture.T he picture was created by Tobias Borries (borriesdesign.de) based on an idea by Edna Bode.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious human pathogen which has been listed as a high-priority multi-drug resistance pathogen by the World Health Organization (WHO). Persistent MRSA infections are often associated with biofilm formation and resistance to conventional antimicrobial therapy. Inhibiting the surface adherence and the virulence of the bacterium is the current alternative approach without affecting growth to reduce the possibility of resistance development. Although numerous antibiofilm agents have been identified, their mode of action remains unclear. Combining two drugs with different modes of action will improve the efficiency of the treatment strategy against MRSA. The present study was aimed to decipher the molecular mechanism underlying the antibiofilm activity of thymol against MRSA and assess the ability of thymol to improve the antibacterial activity of rifampicin. Thymol significantly inhibited 88% of MRSA biofilm formation at 100 µg/ml and reduced the surface adherence of MRSA on glass, stainless steel, and titanium surface coated with human plasma as evidenced by microscopic analyses. qPCR analysis of global virulence regulatory genes and biofilm assay with S. aureus wild type, sarA, and agr strains revealed the sarA-mediated antibiofilm activity of thymol and inhibition of sarA-controlled virulence factors. Congo red assay and erythrocyte lysis assay further confirmed the reduction in polysaccharide intracellular adhesin and hemolysin. Importantly, thymol enhanced the antibacterial and the biofilm eradication efficiency of rifampicin against MRSA and also reduced the formation of persisters. Thus, the present study reveals the sarA-dependent antibiofilm efficacy of MRSA and suggests thymol as the promising combinatorial candidate in potentiating the antibacterial activity of rifampicin against persistent MRSA infections.

A multiplex polymerase chain reaction (PCR) assay has been developed for the identification of the four thermophilic Campylobacter commonly associated with human gastroenteritis including C. jejuni, C. coli, C. lari and C. upsaliensis. The best combination of primers and the annealing temperature of multiplex PCR were examined. Detection limit was 2 ¥ 10 5 cfu and 100 ng of DNA or whole-cell suspension. The multiplex PCR was applied for the direct detection and differentiation of Campylobacter species in 33 human and 45 chicken caeca isolates. Of the 78 specimens evaluated by the multiplex PCR, 55 (70.5%) were identified as C. jejuni, 18 (23.0%) as C. coli and 5 (6.41%) as a mixed infection with both species. Comparison of hippurate test and multiplex PCR demonstrated five (6.41%) isolates with false-positive hippurate enzymic activity and three (3.85%) with false-negative activity. cdtB gene was detected in 100% and 38.9% of C. jejuni and C. coli, respectively.

ABSTRACTCampylobacter hyointestinalisisolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster inC. hyointestinalisisolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015,http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of thecdtgenes inC. hyointestinaliswere found to be significantly different and the gene products are immunologically distinct from those of otherCampylobacterspecies. In this study, we demonstrate the presence of a second variant of thecdtgene cluster inC. hyointestinalis, designatedcdt-II, while the former is namedcdt-I. Sequencing of thecdt-IIgene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB s...

Campylobacter spp., which are gram-negative, highly motile, spiral-shaped bacteria, are important pathogens that cause a variety of diseases in human and animals (10). Of the 17 species currently known in the genus Campylobacter, C. jejuni and C. coli are most frequently isolated from diarrhea patients, particularly children (10). Campylobacter spp. normally resides in the intestinal tract of a variety of wild and domestic animals (10). Among them, poultry has been recognized as the primary reservoir of C. jejuni and C. coli and undercooked chicken represents the most common sources of human Campylobacter infection (10). C. jejuni and C. coli infections share many clinical characteristics but also differs in some manifestations (30). Campylobacter infection is normally self-limiting and treatment

The cytolethal distending toxin (cdt) gene clusters of Campylobacter coli strain Co1-243 and C. fetus strain Col-187 were cloned and sequenced to understand the importance of Cdt as a virulence factor. The cdt genes of C. coli and C. fetus consist of three closely linked genes termed cdtA, cdtB, cdtC whose sizes are 774, 801, and 570 bp, and 702, 798, and 546 bp, respectively. The homologies of each subunit of cdt genes between C. jejuni and C. coli, C. jejuni and C. fetus, or C. coli and C. fetus are 59.6%, 40.3%, or 46.5% for cdtA, 70.2%, 62.4%, or 61.3% for cdtB, 61.3%, 52.3%, or 50.1% for cdtC, respectively. Colony hybridization assay revealed that the genes homologous to the cdtABC gene were distributed in all 27, 19, 20 strains of C. jejuni, C. coli, and C. fetus, respectively, isolated from patients and animals in species-specific manner. Furthermore, nucleotide sequence of the cdt operon, including flanking region, of 10 strains of each species indicated that though the size of the cdtB gene was conserved in each species, those of cdtA and cdtC genes varied particularly among C. coli strains. Amino acid residues demonstrated to be important for toxin activity in CdtB, corresponding to H152, D185, D222, D258, H259 in Cj-CdtB, were also conserved in Cc-CdtB and Cf-CdtB. The cdt gene cluster was located in different sites among different species but in the same site of genomes of the same species. Cdt activity produced by C. jejuni and C. fetus varied among strains, however, any C. coli strains exhibited Cdt activity on HeLa cells. These data indicate that the cdt gene may have a potential for virulence factor at least in C. jejuni and C. fetus.

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacterpositive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases.

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube À1 by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.

Urinary tract infection (UTI) is one of the most common infections in humans. It is primarily caused by uropathogenic Escherichia coli (UPEC), which has a high multidrug resistance (MDR). In consideration of the prevalence of MDR-UPEC strains, the aims of the present study were to systematically review the published data about the prevalence rate of MDR-UPEC from different parts of Iran and to establish the overall relative frequency (RF) of these strains in Iran. We searched several databases including PubMed, ISI Web of Science, Scopus, Google Scholar, IranMedex, and Iranian Scientific Information Database by using the following keywords:``Escherichia coli'',``multidrug resistant'',``MDR'',``urinary tract infections'',``UTI'',``uropathogenic''. and``Iran''. Articles or abstracts that reported the prevalence of MDR-UPEC were included in this review. We found 15 articles suitable for inclusion in this study. A pooled estimation of 10,247 UPEC strains showed that 49.4z (95z confidence interval ＝ 48.0-50.7z) of the stranis were MDR positive. The RF of MDR-UPEC in different studies varied from 10.5z to 79.2z in the Kashan and Hamedan provinces, respectively. According to the results of the present study, the RF of MDR-UPEC in Iran is high. Thus, measures should be taken to keep the emergence and transmission of these strains to a minimum.

Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping. Several enzyme combinations, including the best ones proposed in literature, were utilized and the kigkest discrimination was reached by individual digestion with hull, HindIll, and EcoRl or BamHI. Clinical isolates from outbreaks were clonally related as identified by this molecular approach. Restriction rDNA profiles were composed of strong and weak bands. Using 6 yg DNA we were able to demonstrate that PvulI, HindIII, and BamEII weak bands were reproducible. These weak bands should be consid

Colonization with Pseudmonas cepacia in patients with cystic fibrosis (CF) has been associated with increased morbidity and early death, compared with colonization by P. aeruginosa. The mode of acquisition of P. cepacia is not fully understood, although person-to-person spread appears likely. Recent epidemiologic studies support the importance of social contact in the spread of P. cepacia among patients with CF. This study was undertaken to investigate the epidemiology of P. cepacia colonization among patients with CF attending the CF clinic at our center. Isolates of P. cepacia were collected from patients at two CF treatment centers, including ours. Additional isolates were collected from patients without CF in the hospital ICU, from other teaching hospitals, and from the environment. Profiles of enzymes were obtained by ultrathin polyacrylamide gel electrophoresis of P. cepacia extracts. A predominant electromorphic type (ET) was found among P. cepacia isolates from patients at both centers, suggesting a common source or person-to-person transmission. The majority of hospital isolates fell into a single, different ET. Surveillance swabs of respiratory equipment at our CF clinic did not grow P. cepacia. Attendance of patients at CF summer camp correlated strongly with P. cepacia colonization (P < 0.0001).

Proper surveillance of Campylobacter jejuni and Campylobacter coli, major pathogens associated with human gastroenteritis, is necessary to tackle the increasing disease burden. To detect these pathogenic species, a variety of PCR assays have been developed. This study examined the sensitivity and specificity of 12 PCR assays targeting 23S rRNA, ceuE, lpxA, hipO, mapA, ask, and cdt genes of C. jejuni and C. coli. The sensitivities of PCR assays were 85.2-100%, and 97-100%, and the specificities were 90.5-100%, and 94.3-100% for the tested C. jejuni (n = 61) and C. coli (n = 33) strains, respectively. Two PCR assays, targeting cdtC and hipO genes, were found to be 100% sensitive and/or specific for all C. jejuni strains, while 3 assays, targeting cdtB, cdtA, and ask genes, were 100% sensitive and/or specific for C. coli strains. However, PCR assays for hipO and ask genes are problematic to conduct simultaneously due to the differences in PCR conditions. Overall, multiplex PCR assays targeting cdtC and cdtB genes, encoding 2 subunits of the same toxin, were concluded to be the most reliable. The results of this study would aid in proper surveillance of C. jejuni and C. coli and adopting intervention strategies in the near future.

Resumo: Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes micro-organismos em diferentes habitats tais como água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de Campylobacter jejuni de origem avícola para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido...

The present study evaluated the antibacterial and antibiofilm activity of methanolic extract of Hemidesmus indicus root (MHIR) in combination with amoxicillin and clindamycin against biofilm-forming methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk samples of mastitic cows. Microdilution susceptibility testing and microtitre plate assays were used to evaluate the in vitro efficacy of MHIR and antibiotic combinations against MRSA (n = 12). Furthermore, in vitro findings were validated in a murine model. Minimum inhibitory concentration and minimum biofilm inhibitory concentration of amoxicillin and clindamycin in combination with MHIR were significantly (P &lt; 0·05) lower than when used alone against MRSA. In terms of antibacterial activity, MHIR showed additive interaction (fractional inhibitory concentrationindex &gt;0·5–4) with amoxicillin and clindamycin against all the MRSA isolates, whereas MHIR synergizes (fractional biofilm inhibitory concentrationindex ≤...

Multiresistant Klebsiella pneumoniae strains with plasmid-borne extended-spectrum beta-lactamases (ESBL) are increasingly frequent nosocomial pathogens. A major outbreak of clinical infections, mainly involving patients in the Newborn Services Unit with limited spread to adult patients, occurred at our hospital. This epidemic was investigated by typing the isolates phenotypically and with random amplified polymorphic DNA analysis (RAPD) and plasmid analysis. Forty-eight isolates, consisting of 44 consecutive clinical isolates and 4 selected surveillance isolates, were studied. A single decamer primer was used for the RAPD, and this was effective in demonstrating that the majority of isolates (45 of 48) had the same profile. Three other isolates had different RAPD patterns identifying them as nonepidemic strains. Plasmids were extracted by alkaline lysis with Magic-miniprep kits from 10 isolates selected to represent the epidemic and nonepidemic strains. This method produced small (&...

Background and Objective: Campylobacters are infectious zoonotic agents, and among the main bacterial causes of gastroenteritis in humans. Studies have shown that Campylobacter jejuni is of the main causes of infection among humans. Detection of these infectious agents in water resources is of great importance for maintaining the health of humans. Therefore, the aim of this study was molecular detection of C. jejuni strains in surface water samples collected from Rasht, Iran. Methods: This cross-sectional descriptive study was performed on 45 surface water samples collected from the city of Rasht. After culture and isolation of bacteria, the molecular detection of C. jejuni was carried out using hipO-specific primers. Presence of cytolethal distending toxin (cdt) gene in positive samples was evaluated by polymerase chain reaction using cdtC-specific primers. Results: Of 45 samples, seven (15.5%) were positive for C. jejuni contamination, five of which (71.4%) had the cdtC gene. Conc...

Objective: To study the efflux pump inhibition activity of Mucunacochinchinensis extracts Methods: Sensitivity assay was performed by Kirby Bauer technique. Effluxing ability of microbe was done by accretion, accumulation and efflux pump assays. Results: Resistant fungal strains were subjected to efflux ability studies. Maximum effluxing ability was determined from the experimental data and it was found to be 105 min and 75 min by Candida albicans for methanolic extract of M. cochinchinenesis (MMC) and ethanolic extract of M. cochinchinensis (EMC) respectively and 45 min and 105 min by Aspergillus niger for MMC and EMC respectively. Profound synergistic effect of inhibition was observed with combined MMC and standard Clotrimoxazole when compared to the standard alone; against C. albicansindicates the modulating efficacy of MMC. Conclusion: M. cochinchinensis extracts showed efflux pump modulating activity.

We detected Campylobacter spp. in 5z (20/380) of diarrheal stool samples collected at an outpatient clinic in Kyoto using a commercial loop-mediated isothermal amplification (LAMP) kit with a fluorescent detection reagent after DNA extraction. The sensitivity and specificity were 100z in comparison with those of semi-nested PCR for the differentiation of Campylobacter jejuni and Campylobacter coli. Fourteen of the 20 samples were already determined as C. jejuni by the culture method. All 20 samples were also positive for C. jejuni by the PCR method. Among the 58 cultured samples, the sensitivity of the culture method against the LAMP method was 93.3z (14/15) and the specificity was 100z (43/43). The detection rate of Campylobacter spp. from the heated supernatants by the LAMP method was lower than that from the supernatant after DNA extraction. In total, 25z (5/20) of the Campylobacter-positive samples by the LAMP method were co-infected with norovirus (3/20), rotavirus (1/20), and human parechovirus (1/20), although no other bacterial co-infection was identified by the culture method. C. jejuni was mostly detected in children aged À 5 years throughout the year. Based on these results, we concluded that care should be taken while diagnosing Campylobacter infection in children. Our newly modified LAMP method is a rapid, easy, and useful method for this diagnosis.

The bulbs of Allium species are a known source of antibacterial phytochemicals. Anti-infective, efflux pump and biofilm inhibitory activities of bulb extracts of selected Ghanaian shallots Allium cepa var aggregatum were evaluated using the HT-SPOTi assay and other whole-cell phenotypic screening techniques to determine their possible mechanisms of action. Ethanol and aqueous extracts of white A. cepa inhibited the growth of Mycobacterium smegmatis mc2 155 and Escherichia coli, respectively. The majority of the Allium extracts significantly (p &lt; 0.05) exhibited efflux pump inhibitory activity against all the acid-fast, Gram-positive and Gram-negative strains used. Hexane and chloroform extract of the pink A. cepa and the aqueous extract of the white A. cepa significantly inhibited M. smegmatis biofilm formation. For Pseudomonas aeruginosa, the inhibition was observed at 250 µg/mL for the aqueous extract (~77.34%) and 125 µg/mL for the hexane extract (~76.51%). The results suggest...

The present study was carried out to isolate the organisms present in the skin of female students at the age group of 18-23 from STET Women's College, Sundarakkottai, Mannargudi, Thiruvarur (Dt), Tamilnadu. India. The samples were streak plated onto different agar medium. The colonies thus formed on the plates were identified using Motility, staining and Biochemical tests and confirmed as E. coli, B. subtilis, S. aureus, S. epidermidis and M.luteus by Bergey's Manual of Bacteriology. Totally 46 isolates of bacteria were obtained from 30 skin swab samples. The isolated bacterial pathogens were maintained as pure culture for further studies. Antiseptic and Herbal soaps such as Dettol, Lifebuoy, Margo, and Chandrika were collected from the Cholan super market, Mannargudi, Thiruvarur (Dt), Tamilnadu, India. Isolated organisms were subjected to sensitivity test using 4 different soaps viz Dettol, Lifebuoy, Margo and Chandrika. The antiseptic soap, Dettol showed better response against the bacterial species when compared to others. Dettol was followed by Margo, Chandrika, and Lifebuoy in controlling all the organisms.Totally 46 isolates of bacteria were obtained from 30 skin swab samples.

In 1805, Vieusseux reported the first identification of meningococcal disease [1] while the causative organism, Neisseria meningitidis, was first isolated in 1887 [2]. In 1919 Herrick stated with respect to meningococcal infections "no other infection so quickly slays" [3], a statement that still holds true 80 years later. Despite the advances in high-level intensive care, the mortality and morbidity in patients with meningococcal disease still remains high. The clinical presentation may include a wide spectrum, progressing within a few hours to septic shock and multiorgan system failure (MOP). Swift institution of therapy is of utmost importance, and consists of aggressive cardiovascular and ventilatory support once the diagnosis is suspected and should on no account be delayed. Prevention and control of meningococcal disease, partially achieved by vaccination and chemoprophylaxis, still remains a public health challenge. This chapter summarizes current knowledge of the pathogenesis, therapy, and prevention of meningococcal disease, with emphasis on meningococcal sepsis. I Epidemiology Meningococcal disease occurs worldwide as endemic infections. Its incidence, during the last 30 years, has remained relatively stable at 1-3 per 100000 in most industrialized countries [4], with the majority of cases occurring during the winter and early spring. The highest incidence is in children under the age of 5 years, with a secondary peak during adolescence and early adulthood. There are at least 13 serogroups of N. meningitidis, of which five serogroups, designated A, B, C, Y and W135, account for virtually all disease-causing cases [5]. In Europe, at least 50% of cases are attributable to serogroup B, and most of the remainder to serogroup C, whereas serogroups A and C predominate throughout Asia and Mrica [6]. Currently, in certain areas of the United States, serogroup Y is responsible for approximately one-third of the cases, serogroup C for another onethird, and serogroup B, W135, and uncommon serogroups for the remainder of cases [7]. In all countries, the incidence of group B disease is highest in infants less than a year of age, whereas group C disease frequently causes localized outbreaks amongst teenagers and young adults. Epidemics are usually characterized by a predominance of a single meningococcal serotype, higher incidence rates, and a shift of cases toward older age groups.

Cytolethal distending toxin (CDT)-producing Escherichia coli have been isolated from patients with diarrhea, sepsis and urinary tract infection. CDT of E. coli is divided into five types (CDT-I through CDT-V) based on differences in nucleotide sequences and its genomic location. However, in our recent studies, a few strains of cdt-II gene-positive bacteria, initially identified as atypical E. coli, were re-identified as Escherichia albertii, an emerging enteropathogen, by extensive characterization including multilocus sequence (MLS) analysis and sugar utilization tests. This finding prompted us to investigate if bacteria previously identified as cdt-II gene-positive E. coli might be E. albertii. In the present study, we therefore reexamined the identity of 20 cdt-II gene-positive bacteria isolated from children with diarrhea, which were initially identified as atypical E. coli. By extensive sugar utilization tests, these bacteria showed a closer relatedness to E. albertii than E. coli, because they did not ferment any of the tested sugars including dulcitol, lactose, D-melibiose, L-rhamnose and D-xylose. Further, both phylogenetic analyses based on nucleotide sequences of 7 housekeeping genes (MLS analysis) and rpoB gene showed that all the cdt-II gene-positive bacteria belonged to a distinct lineage of E. albertii from those of E. coli and Shigella boydii. They were also positive by an E. albertii-specific PCR. Taken together, these data suggest that cdt-II gene-positive bacteria previously identified as E. coli are actually E. albertii. Therefore, we suggest a new definition for cdt-II gene-positive E. coli as E. albertii with the inclusion of CDT-II in E. albertii CDT.