Strains in this group were usually negative for the DT104 determi

Strains in this group were usually negative for the DT104 determinant (98%) but positive for the sulfonamides resistance marker (sul1 gene). The class 1 integron marker (intI1) was never detected, though some Group A strains harbored the SGI1 determinant. Moreover, the beta-lactam resistance determinant TEM was present in three strains with A2 profiles. The major genotype A5 accounted

for 67% of Group A strains and was linked to the presence of all four SPI determinants and the plasmid-associated spvC determinant. A second profile, A9, occurred more frequently than the others, accounting for 24% of Group A strains. A5 and A9 genotypes were very Copanlisib clinical trial closely related as the A9 profile shared the A5 determinant profile, differing only by the absence of spvC. Both profiles were encountered every year in strains from various sources (STI571 purchase Figure 1 and Table 2). Group B was the largest, containing 276 strains. The 15 genotypes of Group B were distributed throughout the 10-year study period (1999-2009). The most common genotype was B6, detected in all types of sources and encountered SGC-CBP30 in 76% of Group B strains (n = 210). All determinants except the bla TEM gene were positive in this genotype. The other 14 profiles were much less frequent

(Table 2). Furthermore, 84% of Group B strains were positive for the DT104 marker. Group B strains consistently exhibited sul1 and intI1 determinants, whereas 88% of these strains (n = 244) carried the SGI1 left junction marker. As previously reported, the SGI1 left junction

region was not conserved among all isolates [8]. Atypical profiles were detected in three strains, of which two were isolated from rabbit farms and feces. These 4-Aminobutyrate aminotransferase two strains were negative for the spi_4D determinant located on SPI-4 and assigned to the B14 profile. The third atypical strain, isolated from an eagle, was negative for the ssaQ marker and assigned to the B15 profile (Figure 1). Group C included 49 strains divided into 8 genotypes that were found throughout the study period. All strains from Group C were negative for sul1 marker. They were also negative for intI1 and SGI1 left junction determinants except for two intI1 positive strains (C1 and C3 profiles) isolated either from poultry or swine sources. Likewise, the DT104 marker was rare, observed in only 6.5% (n = 15) of Group C strains (Figure 1). Two other minor groups–D and E–were identified, each composed of a single strain. Genotypes derived from these groups were considered atypical and uncommon. Some SPI virulence genes were missing: ssaQ for the single Group D strain and both mgtC and spi4D for the Group E strain. Group D and E strains were both recovered from environmental samples, suggesting the presence of such atypical isolates in ecosystem niches (Figure 1 and Table 2).

ARMS detected an additional 32 mutations Eighteen of these were

ARMS detected an additional 32 mutations. Eighteen of these were not detected on the sequencing traces and 14 failed to sequence. Three mutations were detected by sequencing only. These were mutations that the ARMS assays were not designed to detect. (B) NSCLC mutations. Eight EGFR mutations were detected in the NSCLC samples by both methods. ARMS detected an additional 10 mutations. Two of these were not analysed

by sequencing as the DNA amount was too low and eight failed to sequence. Nine mutations were detected by sequencing only. These were mutations that the ARMS assays were not designed to detect. Note that there were 27 mutations in 26 patients as one sample was found to contain two mutations. DNA quantity and Blasticidin S supplier ability to detect mutations The first 121 of the melanoma samples yielding DNA were grouped by DNA yield to determine if at low DNA quantity Bindarit supplier the ability to detect mutations was reduced. The groupings (>5 copies, 5-9 copies, 10-49 copies, 50-99 copies, 100-500 copies and >500 copies) were based on the amount of DNA in the control reactions that could be used to estimate the amount of DNA in the sample. There were more groupings at the lower DNA concentration as it was Dactolisib thought that any effect would

be more likely to be observed in these samples. There was no decrease in the ability to detect mutations as the DNA amount decreased. Both DNA sequencing and ARMS gave similar results in each category although overall ARMS detected more mutations. As the DNA concentration increased the number of successful sequencing reactions also increased: at >50 copies per assay input, the analysis success rate was very similar for both ARMS and sequencing. The results are shown in Fig. 2. Figure 2 Mutation detection success on varying the amount of input DNA. The DNA yield was grouped into categories and the percentage of mutations detected calculated for each group. The n values are the successful number of sequencing and ARMS analyses. The

lower yielding samples did not show any decrease in the numbers of BRAF or NRAS mutations detected. Both DNA sequencing and ARMS gave similar results in each category although overall ARMS detected more mutations. As the DNA concentration increased the number of successful sequencing reactions also increased: at >50 copies per assay selleck monoclonal humanized antibody input, the analysis success rate was very similar for both ARMS and sequencing. In some samples at high DNA concentrations (>1000 copies assay input) non-specific signal did occur in the ARMS. In these samples it was important to dilute DNA below 1000 copies per assay input and repeat the analysis. This only affected a minority of samples – most samples in excess of this DNA limit did not exhibit any non-specificity at all. Why this should occur in some samples and not others is not known but adds to the difficulty of analysing FF-PET DNA.

The stained biofilms were visualized by CLSM

The stained biofilms were visualized by CLSM click here with an Olympus FluoView 500 (Olympus Optical Co. Ltd., Japan) microscope. The CLSM used an argon ion laser at 480-490 nm for excitation and a 500-635

nm band pass filter for emission. CLSM images were processed by Olympus FluoView 500 software. Assays were carried out two times. Representative images are presented on Figure 1. Figure 1 Confocal scanning laser microscopy images of biofilm formation on polystyrene, glass microscopic coverslips and cut fragment of silicone urethral PF299804 manufacturer catheters by different bacterial strains: ((A, I, R) Escherichia coli ATCC 25922, (B, J, S) Enterococcus faecalis ATCC 29212, (C, K, T) Enterococcus hirae ATCC 10541, (D, L, U) Candida albicans SC5314) and biofilm inhibition after incubation with pseudofactin II (0.25 mg/ml) in the culture medium: (E, M, W) Escherichia coli ATCC 25922, (F, N, X) Enterococcus faecalis ATCC 29212, (G, O, Y) Enterococcus hirae ATCC 10541, (H, P, Z) Candida albicans mμSC5314). Scale bars: 50 μl. Biofilm formation in urethral catheters The uropathogenic strains E. coli, E. faecalis, E. hirae and C. albicans were used in these tests. Ten microliter

volumes of overnight cultures of E. coli ATCC 25922, E. faecalis ATCC 29212, E. hirae ATCC 10541 were added into 1000 μl of fresh LB medium, and the same volume of C. albicans SC5314 was added into 1000 μl of fresh RPMI-1640 medium. To the medium was added 1000 μl pseudofactin II (final concentration 0.25 mg/ml) solution in LB medium (for bacterial) and RPMI-1640 medium for C. albicans

selleck chemicals llc and 4 cm long segments of sterile silicone urethral catheters (Unomedical, Denmark). The catheters were incubated at 37°C overnight. The cultures were removed and the catheters Depsipeptide were washed with distilled water. After washing, 3000 μl of crystal violet (0.1%) was added to the catheters for 20 min. The stained biofilms were rinsed three times with distilled water and allowed to dry at room temperature for 15 min before examination. In a parallel experiment the catheters were pretreated with pseudofactin II by being placed in a tube with 2000 μl of 0.25 mg/ml pseudofactin II dissolved in PBS, incubated for 2 h at 37°C and subsequently washed twice with PBS. Then the experiment was carried out as in the case of adding pseudofactin II into the growth medium. Assays were carried out two times. Representative images are presented on Figure 2. This experiment was carried out under dynamic conditions using a peristaltic pump, where the flow of culture with or without pseudofactin II trough urethral catheters was 50 ml/h. Figure 2 Pseudofactin II inhibits biofilm formation on silicone urethral catheters. The organisms were grown overnight at 37°C in a test-tube with sterile urethral catheters containing medium (A) with and without 0.25 mg/ml pseudofactin II and (B) where the urethral catheters was pre-incubated with biosurfactant at concentration 0.25 mg/ml as described in the text.

Results and discussion PspA families and clade distribution Among

Results and discussion PspA families and clade distribution Among the 112 pneumococci studied, the majority (59.8%, 67/112) were identified as belonging to PspA family 2 (31 isolates of clade 3, 27 of clade 4 and nine of clade 5), while the remaining 39.3% (44/112) belonged to family 1 (29

isolates of clade 1 and 15 of clade 2). One strain was negative. No PspA family 3 isolates were detected. Figure 1 shows the phylogenetic tree of the 27 new PspA sequences found as well as the accession numbers and the percentage of identity to LXH254 manufacturer previously published sequences. Sequences of Trichostatin A in vitro strains of PspA families 1 and 2 were precisely grouped, and all were joined into their respective clades. The similarity of isolates of the same family ranged from 84% to 100%. The percentage of similarity within isolates of the same clade ranged as follows: clade 1 (84 to 95), clade 2 (84 to 100), clade 3 (93 to 99), clade 4 (91 to 98) and clade 5 (96 to 100). Among the 66 pneumococci isolated from patients with IPD, 63,6% (42/66) were found to be of PspA family 2 (24 isolates of clade 3, 12 of clade 4 and six of clade 5), 34.8% (23/66) of family 1 (20 isolates of clade 1 and three

of clade 2) and one isolate was negative. The high prevalence of PspA family 2 among pneumococci MEK162 concentration isolated from adults with IPD has already been

reported in Spain, Canada, Sweden, the USA and France [37, 38], although in Australia, the UK and Japan PspA family 1 was the Decitabine order most prevalent [38, 39]. The dominance of family 2, clade 3 observed in our study has also been reported in other studies of pneumococci causing IPD in adults in France [37] and in children from Germany [40]. PspA family 2 was also dominant (54.3%, 25/46) among pneumococci isolated from the nasopharynx of healthy children (seven of clade 3, 15 of clade 4 and three of clade 5), while family 1 accounted for 45.7% (21/46) of the strains (nine of clade 1 and 12 of clade 2). These data are in agreement with two PspA studies [32, 34] which found PspA family 2 to be dominant among pneumococci isolated from Brazilian children carriers. Moreover, the clade distribution also showed a prevalence of clade 4, followed by clade 1 and clade 3 [34]. A recent publication with data collected from pneumococci isolated from nasopharyngeal carriage in Finnish children showed similar prevalences of PspA family 1 and family 2 [41].

Structural elements are in capital letters with the name of the c

Structural elements are in capital letters with the name of the corresponding feature underneath them. Underlined and in italics:

possible transmembrane helix. In bold and italics: alpha helices. Underlined: Beta-sheets. In white letters and highlighted in black: meander loop and Cys pocket. The asterisks (*) indicate the three totally conserved amino acids among cytochromes P450, and the exclamation points (!) show the amino acid variation found in the deduced CYP61 from different X. dendrorhous strains. The CYP61 gene mutation To study the function of the CYP61 gene in X. dendrorhous, mutant cyp61 – strains were generated. The wild-type strains UCD 67–385 and CBS 6938 were transformed with plasmid pBS-cyp61/Hyg, and strain AVHN2 was transformed MK-0518 with plasmid pBS-cyp61/Zeo. All transformations were performed with linearized plasmids as indicated in Figure  4. Through a double homologous recombination event, the donor DNA fragment containing the CYP61 gene JPH203 interrupted by one of the two resistance markers replaced the CYP61 gene in the yeast chromosome. In this way, we obtained the transformant strains 385-cyp61 hph , CBS-cyp61 hph and Av2-cyp61 zeo (Table  2). The learn more genotype modifications in the transformant strains were validated

by PCR reactions using specific primers for the CYP61 gene, zeocin or hygromycin B resistance cassettes (Table  1) and genomic DNA from the parental and transformant strains. The amplicons confirmed the CYP61 gene interruption (Figure  5). However, as strain UCD 67–385 is diploid [30] and we were able to detect a CYP61 wild-type allele, the resulting strain 385-cyp61 hph is heterozygous (385-CYP61/cyp61 hph ). For this reason, strain 385-CYP61/cyp61 hph was transformed with the linearized plasmid pBS-cyp61/Zeo obtaining the cyp61 – homozygote mutant strain 385-cyp61 hph find more /cyp61 zeo (Figure  5). The ploidy levels of strains CBS 6938 and AVHN2 are unknown; based on random mutagenesis experiments

and by transformation of carotenogenic genes performed at our laboratory [21, 31], we estimate that these strains are aneuploid. In these cases, the PCR-based genotype analysis determined that a unique CYP61 gene copy was mutated in strains CBS-cyp61 hph and Av2-cyp61 zeo (Figure  5), indicating that these strains are hemizygous, so a second transformation event was not necessary in these mutants. Interestingly, a clear difference in the color phenotype could be distinguished among all the cyp61 – mutants and their corresponding parental strains, indicating alterations in carotenoid biosynthesis (see below). Figure 4 Plasmids constructed in this work. In each plasmid illustration, relevant features for this work, such as endonuclease recognition sites and primer binding sites (thin arrows), are shown. Some elements of the original plasmid (pBluescript SK-) were kept and shown in gray. Plasmid pBS-gCyp61 harbors the genomic version of the CYP61 gene from X.

PubMedCrossRef 2 Erwin AL, VanDevanter DR: The Pseudomonas aerug

PubMedCrossRef 2. Erwin AL, VanDevanter DR: The Pseudomonas aeruginosa genome: how do we use it to develop strategies for the treatment of patients with cystic fibrosis and Pseudomonas infections? Curr Opin Pulm Med 2002,8(6):547–551.PubMedCrossRef 3. Richards MJ, Edwards JR, Culver DH, Gaynes RP: Nosocomial infections Geneticin solubility dmso in medical intensive care units in the United States.

National Nosocomial Infections Surveillance System. Crit Care Med 1999,27(5):887–892.PubMedCrossRef 4. Elkin S, Geddes D: Pseudomonal infection in cystic fibrosis: the battle continues. Expert Rev Anti Infect Ther 2003,1(4):609–618.PubMedCrossRef 5. Zhang L, click here Parente J, Harris SM, Woods DE, Hancock RE, Falla TJ: Antimicrobial peptide therapeutics for cystic fibrosis. Antimicrob Agents Chemother 2005,49(7):2921–2927.PubMedCrossRef 6. Kipnis E, Sawa T, Wiener-Kronish J: Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect 2006,36(2):78–91.PubMedCrossRef 7. check details Murray TS, Egan M, Kazmierczak BI: Pseudomonas aeruginosa chronic colonization in cystic fibrosis patients. Curr Opin Pediatr 2007,19(1):83–88.PubMedCrossRef 8. Hentzer M, Teitzel GM, Balzer GJ, Heydorn A, Molin S, Givskov M, Parsek MR: Alginate overproduction affects Pseudomonas

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Lung Cell Mol Physiol 2000,279(5):L799–805.PubMed 14. Devine DA: Antimicrobial peptides in defence of the oral and respiratory tracts. Mol Immunol 2003,40(7):431–443.PubMedCrossRef 15. Zhang L, Falla TJ: Cationic antimicrobial peptides – an update. Expert Opin Investig Drugs 2004,13(2):97–106.PubMedCrossRef 16. Toke O: Antimicrobial peptides: new candidates in the fight against bacterial infections. Biopolymers 2005,80(6):717–735.PubMedCrossRef 17. De Smet K, Contreras R: Human antimicrobial peptides: defensins, cathelicidins and histatins. Biotechnol Lett 2005,27(18):1337–1347.PubMedCrossRef 18. Zhang L, Falla TJ: Antimicrobial peptides: therapeutic potential. Expert Opin Pharmacother 2006,7(6):653–663.PubMedCrossRef 19. Hale JD, Hancock RE: Alternative mechanisms of action of cationic antimicrobial peptides on bacteria.

Potential binding sequence of AirR was listed below (PDF 225 KB)

Potential binding sequence of AirR was listed below. (PDF 225 KB) Additional file 4: Comparison of microarray result of previous report. The table contains both microarray data and the verification result of real-time RT PCR. (PDF 108 KB) References 1. Lowy FD: Staphylococcus aureus infections. N Engl J Med 1998,339(8):520–532.selleck products PubMedCrossRef 2. Diep BA, Otto M: The role of virulence determinants c-Met inhibitor in community-associated MRSA pathogenesis.

Trends Microbiol 2008,16(8):361–369.PubMedCentralPubMedCrossRef 3. Hiramatsu K: Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis 2001,1(3):147–155.PubMedCrossRef 4. O’Riordan K, Lee JC: Staphylococcus aureus capsular polysaccharides. Clin Microbiol Rev 2004,17(1):218–234.PubMedCentralPubMedCrossRef 5. Stock AM, Robinson VL, Goudreau PN: Two-component signal transduction.

Annu Rev Biochem 2000, 69:183–215.PubMedCrossRef 6. Queck SY, Jameson-Lee M, Villaruz buy GDC-0941 AE, Bach TH, Khan BA, Sturdevant DE, Ricklefs SM, Li M, Otto M: RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol Cell 2008,32(1):150–158.PubMedCentralPubMedCrossRef 7. Novick RP: Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 2003,48(6):1429–1449.PubMedCrossRef 8. Li D, Cheung A: Repression of hla by rot is dependent on sae in Staphylococcus aureus. Infect Immun 2008,76(3):1068–1075.PubMedCentralPubMedCrossRef 9. Toledo-Arana A, Merino N, Vergara-Irigaray M, Debarbouille M, Penades JR, Lasa I: Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system. J Bacteriol 2005,187(15):5318–5329.PubMedCentralPubMedCrossRef 10. Brunskill EW, Bayles KW: Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus Carnitine palmitoyltransferase II aureus. J Bacteriol 1996,178(3):611–618.PubMedCentralPubMed 11. Torres VJ, Stauff DL,

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FEBS J 2005, 272:1326–1342 PubMedCrossRef 36 Hawkins CF, Borges

FEBS J 2005, 272:1326–1342.PubMedCrossRef 36. Hawkins CF, Borges A, Perham RN: A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett 1989, 255:77–82.PubMedCrossRef 37. Meshalkina L, Nilsson U, Wikner C, Kostikowa T, Schneider G: Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis. Eur J Biochem 1997, 244:646–652.PubMedCrossRef 38. Abedinia M, Layfield R, Jones SM, Nixon PF, Mattick JS: Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase. Biochem Biophys Res Commun 1992, 183:1159–1166.PubMedCrossRef 39. Jakobsen OM, Brautaset T, Degnes KF, Heggeset TM, Balzer S, Flickinger MC,

Valla S, Ellingsen TE: Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus. Appl Environ Microbiol

2009, 75:652–661.PubMedCentralPubMedCrossRef 40. Kelley-Loughnane N, Biolsi SA, Gibson KM, Lu G, Hehir MJ, Phelan P, Kantrowitz ER: Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase. Biochim Biophys Acta 2002, 1594:6–16.PubMedCrossRef 41. Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF: Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperature-triggered glutamate production. Appl Environ Microbiol 2005, 71:5920–5928.PubMedCentralPubMedCrossRef 42. Haima P, van Sinderen D, Bron S, Venema CB-839 nmr G: An improved beta-galactosidase alpha-complementation system for molecular cloning in Bacillus subtilis . Gene 1990, 93:41–47.PubMedCrossRef 43. Brautaset T, Jakobsen OM, Degnes KF, Netzer R, Naerdal I, Krog A, Dillingham R, Flickinger MC, Ellingsen TE: Bacillus methanolicus

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With the highest value of VC, the state variable can be in SET st

With the highest value of VC, the state variable can be in SET state, where the emulator circuit can be considered a SET resistance. Figure 2c shows the

voltage waveform of V C with respect to time. At the starting point of sinusoidal function of V IN, V C is 1.2 V that is decided by D1 in Figure 1. After the half cycle of sinusoidal function, V C reaches 2.8 V. When one cycle of sinusoidal function is completed, the V C value returns to the value at the starting point of sinusoidal function. Figure 2d shows a typical pinched hysteresis loop of a memristor’s voltage and VX-680 mw current which are emulated by the proposed circuit in Figure 1. In the simulation, V DD is 3.3 V and the frequency of sinusoidal function is 10 kHz. Figure 2 Simulated voltage waveforms. The simulated voltage waveforms of (a) see more V IN, (b) I IN, (c) V C, and (d) the pinched hysteresis loop

of the voltage-current relationship GSK2126458 datasheet of the proposed emulator circuit when the sinusoidal frequency is 10 kHz. The simulated voltage waveforms of (e) V IN, (f) I IN, (g) V C, and (h) the pinched hysteresis loop of the voltage-current relationship of the proposed emulator circuit when the sinusoidal frequency is 40 kHz. Figure 2e, f, g, h shows the simulation results of the proposed emulator circuit with four times higher frequency of 40 kHz than that of Figure 2a, b, c, d, V IN, I IN, V C, and the pinched hysteresis loop, respectively, with 10 kHz. A sinusoidal voltage with 40 kHz that is applied to the emulator circuit is shown in Figure 2e. Here the first three peaks are for increasing V C in Figure 1; thereby, the emulator circuit changes from RESET to SET. The next three peaks are for decreasing the state variable; thus, the emulator circuit can return

to RESET. I IN and V C with the sinusoidal function that is indicated in Figure 2e are shown in Figure 2f, g, respectively. Figure 2h shows the voltage-current Florfenicol relationship of the emulator circuit. In Figure 2h we can see three voltage-current loops at the right and another three voltage-current loops at the left which correspond to the three high peaks and three low peaks in Figure 2e, respectively. Figure 3a shows SET pulses with different amplitude values. Here the amplitude values are increasing monotonically from 0.5 to 3 V. Each SET pulse is followed by a RESET pulse with the fixed amplitude as high as 3 V that is shown in Figure 3b. The state variable that is changed by SET and RESET pulses are shown in Figure 3c. Here V C represents the amount of stored charge at C1 that controls the voltage-controlled resistor in Figure 1 that acts as memristor. Figure 4a shows the read and write circuits for the proposed emulator circuit of memristors [9, 10]. The read circuit is simply composed of a current mirror and comparator. The comparator G1 compares the sensing voltage V SEN with the reference voltage V REF.

​ncbi ​nlm ​nih ​gov) and subsequently aligned to the sequence of

​ncbi.​nlm.​nih.​gov) and subsequently aligned to the sequence of the reference plasmid, pUTI89 [GenBank:CP000244]. Gap closure was performed using primer walking into the gaps with

the LongRange PCR Kit (Qiagen). ON-01910 research buy The complete sequence of the plasmid was annotated using Rapid Annotation using Angiogenesis inhibitor Subsystem Technology (RAST) [34]. Comparative genomics and phylogenetic analysis Comparative genomics of pRS218 with closely related IncFIB/FIIA plasmids of other E. coli was performed using Mauve 3.2.1 genome alignment web tool (http://​gel.​ahabs.​wisc.​edu/​mauve/​) [35]. An evolutionary relationship of 24 plasmids belonging to the IncFIB/FIIA group based on repA1 gene sequence was performed using the neighbor-joining method. A neighbor joining tree was constructed by using the MEGA4 web tool (http://​www.​megasoftware.​net/​mega4/​mega.​html) [36,37]. Analysis of plasmid profiles of NMEC strains Extraction of large plasmids from NMEC strains was performed using an alkaline lysis method described previously [33]. In brief, 1 ml of overnight culture of each E. coli strain was subjected to alkaline lysis using 10% sodium hydroxide followed by phenol-chloroform

extraction of plasmid DNA. Plasmid BMS202 profiles of NMEC strains

were evaluated by electrophoresis on a 0.7% agarose gel containing 0.5 μg/ml ethidium bromide. Evaluation of prevalence of selected pRS218 genes in other NMEC and fecal E. coli Specific polymerase chain reactions (-)-p-Bromotetramisole Oxalate (PCRs) were performed to determine the presence of selected gene coding regions (n = 59) of pRS218 in other NMEC and fecal E. coli strains. Primers were designed using the Primer 3.0 web tool (http://​bioinfo.​ut.​ee/​primer3-0.​4.​0/​) (Table 5). PCR amplifications were performed using crude DNA extracted by the rapid boiling method [38]. The PCR mixture contained 1 U of Taq polymerase (Qiagen), 1× Taq polymerase buffer, 3.5 mM MgCl2, 125 μM each deoxynucleotide triphosphate (dNTP) and150 nM each primer pair. PCR conditions were as follows: 1 cycle of 95°C for 1min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 10 min. Amplicons were visualized on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Table 5 Primers used for the screening of pRS218 genes among neonatal meningitis causing E. coli and fecal commensal E.