Triplex Polymerase Chain Reaction Research Articles

Development and Application of a Triplex PCR Assay for Detection of Koi Herpes Virus (KHV) Gene Infected Common Carp (Cyprinus carpio L.)

Koi herpesvirus (KHV) has been responsible for worldwide KHV disease (KHVD) outbreaks, resulting in high mortality rates, and has been reported in Cyprinus carpio cultivation. A Triplex Polymerase Chain Reaction (T-PCR) was developed for the detection of KHV gene to improve the diagnosis of KHV infection. Three sets of primers were designed targeting specific sequences of KHV gene which were frequently used in PCR assay for detecting KHV (modifications primer by Yuasa,290 bp; Gilad 484 bp, and TK, 409 bp) in once PCR running. The validity test was conducted by verified using DNA sample of Aeromonas hydrophila. Sensitivity test of singleplex and Triplex conducted by dilution. The result showed that T-PCR method using these pairs of primers is at 55 annealing temperature with consistency of T-PCR method has been repeated in triplicates, and with the same results. The validity test found that no band detected with A. hydrophila. This Triplex PCR-based assay may have useful for the clinical diagnosis tool of KHV disease.

Quality improvement study on the effectiveness of intranasal povidone-iodine decolonization on surgery patients

Surgical site infection prevention and treatment remains a challenge in healthcare settings globally. The routine use of intranasal mupirocin for decolonization has challenges and preoperative intranasal povidone-iodine decolonization is another option. The purpose of this quality improvement study was to assess if a one-time preoperative intranasal povidone-iodine application could reduce the risk of the likelihood of nasal carriage of Staphylococcus aureus after surgery. Ambulatory Surgery Center patients were enrolled in an intranasal povidone-iodine decolonization quality improvement study as they reported at the pre-operative holding area. Pre-decolonization intranasal samples were collected, followed by intranasal application of povidone-iodine. Patients waited for a minimum of 20 minutes after application before proceeding with surgery. Nasal samples were again collected after surgery. Each sample was tested for S.aureus colonization using the 16S rRNA-mecA-nuc triplex polymerase chain reaction, standard biochemical tests, and qualitative culturing. In the 98 patients enrolled, 36% of these patients had intranasal colonization with S.aureus by 16S rRNA-mecA-nuc triplex polymerase chain reaction before surgery. Using a qualitative culture technique, 28% of patients tested positive for S.aureus before surgery and 20% of patients tested positive for S.aureus after surgery (P= 0.039). Intranasal preoperative povidone-iodine is an effective strategy in the decolonization of S.aureus from the nares if properly implemented.

Virulence Genotyping and Multidrug Resistance Pattern of Escherichia coli Isolated From Community-Acquired and Hospital-Acquired Urinary Tract Infections.

IntroductionUropathogenic Escherichia coli(UPEC) strains consist of a plethora of putative virulence factors (VFs), which help them to establish infection in the urinary tract. We compared genotypic profiles of Escherichia coli (E. coli) strains associated with community-acquired (CA) urinary tract infection (UTI; n=100) and hospital-acquired (HA) UTI (n=50) in the present study in order to identify specific virulence determinants, if any, associated with either form of UTI and its association with antibiotic resistance pattern of the isolates.Materials and methodsE. coli strains were analyzed for antimicrobial susceptibility patterns, phylogroups, and 10 putative virulence-associated genes. The bacterial culture and identification were done using standard conventional methods. Tests for antimicrobial susceptibility and phenotypic detection for extende- spectrum beta-lactamases (ESBL) were done by using the Kirby Bauer disc diffusion method, and results were interpreted as per Clinical & Laboratory Standards Institute (CLSI) guidelines. The phylotype (A, B1, B2, and D) of each E. coli isolate was determined by a triplex polymerase chain reaction (PCR) based phylotyping method. They were further analyzed for the presence of 10 putative virulence genes (VGs), including adhesins papA (P fimbrial structural subunit), papG alleles I, II (P fimbrial adhesin variants), fimH (type 1 fimbriae), toxins hlyA (hemolysin) siderophores chuA (heme-binding protein); yfcV (encodes a major subunit of a putative chaperone-usher fimbria) capsule synthesis specific for group II (K1, K5, K12, etc.) kpsMII; serum resistance‐associated traT, and upaH by multiplex PCR.ResultsHA E. coli isolates were significantly more drug-resistant than CA isolates; carbapenem (80% vs. 16%), ceftazidime (92% vs. 63%). The majority (52%) of E.coli isolates associated with HA UTI belong to commensal phylogroup A and B1, whereas the majority (66%) in CA were from pathotypic phylogroups, i.e., B2 & D. Most of VFs were frequently present amongst CA group except for traT and yfc, kpsMTII, hlyA, chuA, and upaH were significantly associated with CA E.coli isolates while yfc was significantly present in HA E.coli isolates. Though adhesin genes such as papA, papGI, papGII, fimH were frequently found in the CA group, they were not significantly associated. The average virulence score was higher for CA UTI isolates (4.25) than for the HA strains (3.9). Multidrug resistance (MDR) was present in every HA E.coli isolate, and fimH, traT, and yfc genes showed significant association with MDR strains.ConclusionOn detailed analysis, we found that HA E. coli isolates had a high frequency of MDR and comparatively reduced VFs content. Thus, it can be assumed that a strain with lesser virulence is able to cause HA UTIs, as compared to CA UTIs, which probably indicates that the host’s immune status/general condition can be an important determinant in acquiring infection rather than virulence potential of the pathogen alone.

Phylogenetic groups and antimicrobial resistance characteristics of Escherichia coli strains isolated from clinical samples in North Iran

Background and study aimExtraintestinal pathogenic Escherichia coli (ExPEC) is one of the most common bacterial pathogens, which causes a remarkable amount of morbidity and mortality. This study was designed to determine the antibiotic resistance profiles, phylogenetic groups, and subgroup analyses among the ExPEC strains isolated from hospitalized patients in north Iran. Patients and MethodsThis cross-sectional investigation was conducted at five educational hospitals in Rasht in north Iran. Using standard microbiological tests, 150 E. coli isolates were identified. The antibiotic susceptibility pattern of all isolates was determined using the disk diffusion method. The double disk phenotypic confirmatory test was performed to detect extended-spectrum β-lactamase (ESBL)-producing isolates. A triplex polymerase chain reaction (PCR) was performed to determine the phylogenetic group of each strain. ResultsThe results of antibiogram pattern showed that E. coli isolates were mostly non-susceptible to ampicillin (79.3%), followed by nalidixic acid (75.3%) and cephalothin (70%), whereas nitrofurantoin (94.7%) was the most effective agent, followed by imipenem (92.7%). The rate of ESBL-producing isolates was 53.3% (80/150). Multiplex PCR screening revealed that the most common phylogroup was the B2 group (97 isolates; 64.6%), followed by the D group (34, 22.7%). In contrast, phylogroup analyses showed that B23 (50.7%) and D2 (16.4%) were the most common subgroups. ConclusionsOur findings indicated a considerable rate of antibiotic resistance and ESBL-producing isolates among E. coli strains isolated from clinical samples. Moreover, we reported a tendency that most isolates belonged to the B2 and D phylogroups. As a result, the detection of genotypic identical or similar isolates indicated that these isolates have an endurance capability in the hospital environment and could be transmitted among patients.

Development of triplex PCR for simultaneous detection of soybean and wheat.

There is a constant demand for an effective detection method of major allergens, such as soybean (Glycine max) and wheat (Triticum aestivum). In this study, a triplex polymerase chain reaction (PCR) method was developed for rapid detection of soybean and wheat in processed food products. This triplex PCR contains soybean- and wheat-specific primer pairs and universal primer pair for plant species. Each primer pair was applied to 22 different plant species and showed high specificity with no amplification in non-target species. The sensitivity of triplex PCR for soybean and wheat was 10pg. The detection limit for soybean and wheat in pea mixture was 0.1%. The developed triplex PCR showed high sensitivity and specificity and was applied to 21 different commercial products. The results were in accordance with the label. Thus, this method is expected to be useful in preventing allergy-related issues via accurate labeling of major allergens in food.

Prevalence of urogenital, anal, and pharyngeal infections with Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium: a cross-sectional study in Reunion island

BackgroundRecommendations for sexually transmitted infection (STI) screening vary significantly across countries. This study evaluated the prevalence of urogenital and extragenital infections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Mycoplasma genitalium (MG) in patients visiting a French STI clinic in the Indian Ocean region to determine whether current STI screening practices should be updated.MethodsThis cross-sectional study examined all patients who visited the STI clinic between 2014 and 2015. Triplex polymerase chain reaction screening for CT, NG, and MG was performed on urine, vaginal, pharyngeal, and anal specimens (FTD Urethritis Basic Kit, Fast Track Diagnostics, Luxembourg).ResultsOf the 851 patients enrolled in the study, 367 were women (367/851, 43.2%) and 484 were men (484/851, 56.0%). Overall, 826 urogenital specimens (826/851, 97.1%), 606 pharyngeal specimens (606/851, 71.2%), and 127 anal specimens (127/851, 14.9%) were taken from enrolled patients. The prevalence of urogenital CT and MG was high in women ≤25 years (19/186, 10.21%; 5/186, 2.69%) and in men who have sex with women ≤30 years (16/212, 7.54%; 5/212, 2.36%). Among patients with urogenital CT infection, 13.7% (7/51) had urethritis. All patients with urogenital MG infection were asymptomatic. Men who have sex with men had a high prevalence of pharyngeal CT (2/45, 4.44%) and NG (3/44, 6.81%) and a high prevalence of anal CT (2/27, 7.41%), NG (2/27, 7.40%), and MG (1/27, 3.70%). After excluding patients with concomitant urogenital infection, extragenital infections with at least 1 of the 3 pathogens were found in 20 swabs (20/91, 21.9%) taken from 16 patients (16/81, 19.7%), all of them asymptomatic.ConclusionsRoutine multisite screening for CT, NG, and MG should be performed to mitigate the transmission of STIs in high-risk sexually active populations.

Aeromonas hydrophila, one reason causing the death of freshwater crayfish Procambarus clarkii (Girard, 1852)

Procambarus clarkii crayfish has been developed as an excellent animal model to study the characteristics and pathogenesis of white spot syndrome virus and the host defense mechanisms of crustacean. However, due to pathogen infections of crayfish in Chinese farms, research based on this model has been slowed down. Using triplex polymerase chain reaction, we found the virulence Aeromonas hydrophila was one reason that caused the death of crayfish. The isolated AH-3 produced 100% mortalities with 3-24h after inoculation of 1×106 colony-forming units of live cells per crayfish at 20 ˚C.

High Resolution Melt Assays to Detect and Identify Vibrio parahaemolyticus, Bacillus cereus, Escherichia coli, and Clostridioides difficile Bacteria.

Over one hundred bacterial species have been determined to comprise the human microbiota in a healthy individual. Bacteria including Escherichia coli, Bacillus cereus, Clostridioides difficile, and Vibrio parahaemolyticus are found inside of the human body and B. cereus and E. coli are also found on the skin. These bacteria can act as human pathogens upon ingestion of contaminated food or water, if they enter an open wound, or antibiotics, and environment or stress can alter the microbiome. In this study, we present new polymerase chain reaction (PCR) high-resolution melt (HRM) assays to detect and identify the above microorganisms. Amplified DNA from C. difficile, E. coli, B. cereus, and V. parahaemolyticus melted at 80.37 ± 0.45 °C, 82.15 ± 0.37 °C, 84.43 ± 0.50 °C, and 86.74 ± 0.65 °C, respectively. A triplex PCR assay was developed to simultaneously detect and identify E. coli, B. cereus, and V. parahaemolyticus, and cultured microorganisms were successfully amplified, detected, and identified. The assays demonstrated sensitivity, specificity, reproducibility, and robustness in testing.

Prevalence and characterization of Escherichia coli in the Kelantan River and its adjacent coastal waters

AbstractThe presence of Escherichia coli in river and sea water may cause different levels of infections and constitutes a risk to public health. In this study, water samples were collected from 15 sites along the Kelantan River, estuaries and its adjacent coastal waters to investigate the prevalence and diversity of E. coli. A membrane filtration technique was used to enumerate E. coli and phylogenetic grouping was performed using triplex polymerase chain reaction. E. coli abundance ranged from 3.1 × 10 to 1.6 × 105 colony forming units 100 mL−1, and total suspended solids correlated significantly with E. coli abundance (r2 = 0.165, p < 0.001) and rainfall (r2 = 0.342, p < 0.001). Phylogenetic group B1 and A (59.4%) were the most prevalent, whereas groups B2 and D were least abundant. The higher abundance of phylogenetic group D at upstream sites of the Kelantan River suggested fecal contamination mainly of animal origin. Canonical-correlation analysis showed phylogenetic group B2, and phylogenetic groups A and D were greater in waters with higher inorganic nutrients (e.g. NH4, NO2 and NO3), whereas phylogenetic group B1 appeared to have better salinity tolerance between phylogenetic groups.

Molecular Detection, Virulence Genes, Biofilm Formation, and Antibiotic Resistance of Salmonella enterica Serotype enteritidis Isolated from Poultry and Clinical Samples

Background: Salmonella spp. is one of the most important zoonotic pathogens transmitting among human and animals. Due to the similarity of antibiotic classes used to treat animals and humans, there is a high risk for emerging the multi-drug resistant (MDR) strains. Objectives: The current study aimed at evaluating molecular detection, virulence genes, biofilm formation, and antibiotic resistance of Salmonella enterica serotype enteritidis recovered from poultry and clinical isolates. Methods: A total of 282 isolates were recovered from chicken meat, live poultry feces, eggs, and human feces in Iran. The presence of virulent factors in the isolates was confirmed using biochemical and microbiological tests. The presence of Salmonella genus was determined using antiserum. Triplex polymerase chain reaction (PCR) was performed to detect Salmonella spp., serogroup D and the discriminate S. enteritidis from other species. Kirby-Bauer disk diffusion method was applied to perform the susceptibility testing. Quantification of biofilm formation was determined in 96-well microtiter plates as recommended by the defined protocol. The data were then analyzed with SPSS using consensus tables and Chi-square test. Results: Based on the results, all the isolates were positive for invA, sdiA, hilA, and ratA. Moreover, spvC had the lowest prevalence (37.6). Of all strains, 67 were MDR, 51.7 of which were recovered from humans. Furthermore, 34.5 of isolates were strong biofilm producers. There was a significant correlation between the strong biofilm formation and the antibiotic resistance to colistin, ceftazidime, chloramphenicol, gentamicin, trimethoprim, penicillin, and trimethoprim-sulfamethoxazole. Conclusions: The results of the current study showed a significant correlation between the strong biofilm formation and the antibiotic resistance to some antibiotics. © 2018, Author(s).

Molecular description of alpha-hemolysin producing Escherichia coli isolates from extraintestinal source

Context: Alpha-hemolysin (Hly) is a common exotoxin produced by Escherichia coli, which plays an important role as a virulence factor in a number of clinical infections. There exist other virulence properties exhibited by such strains whose role in pathogenesis have not yet been clearly elucidated. Aims: Aim of present study was to characterize those alpha-hemolysin producing isolates on the basis of their virulence factors and phylogenetic background. Settings and Design: This descriptive study was carried out in a multi-specialty tertiary care hospital. Methods and Material: Three hundred non-repeat E. coli isolates from in-patients were studied. Isolates were differentiated as haemolytic and non-haemolytic on the basis of both phenotypic methods such as haemolysis on sheep blood agar and genotypic detection of the hly gene. Possession of virulence and drug resistance genes was determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiotic sensitivity testing was performed by disk diffusion method. Results: Of the 300 isolates, it was observed that by phenotypic (haemolysis on blood agar) method, 65(22%) were hemolytic whereas the genotypic method detected 70 (23.5%) isolates possessing alpha haemolysin (hlyA+) marker. B2 phylogroup isolates were found to harbour the hlyA+ marker at a significantly higher (P < 0.05) rate.  The possession of papC and cnf1 genes was significantly higher in hlyA+ isolates. Conclusions: Alpha-haemolysin producing isolates demonstrated higher virulence properties. The microbiology laboratory may report haemolytic characteristics of the isolates as it may alert the physician of the higher pathogenic potential of the strain and to initiate appropriate treatment.

Salmonella Gallinarum field isolates and its relationship to vaccine strain SG9R

ABSTRACT1. The aim of the present study was to determine if the 9R-strain of the Salmonella Gallinarum live vaccine was responsible for having fowl typhoid outbreaks in chicken flocks from both chicken and turkey breeders as well as to verify the antimicrobial resistance of the isolates from the outbreaks.2. The triplex polymerase chain reaction, standard antimicrobial test, beta-lactamase genes identification and Ion Torrent PMG whole-genome sequence were used in the field isolates and in the vaccine strain of S. Gallinarum.3. The 60 tested isolates were not from vaccine origin and manifested high resistance to drugs from macrolide and quinolone groups. Whole-genome sequencing (WGS) and single nucleotide polymorphism analysis on selected isolates for core genes from Salmonella enterica confirmed the wild origin of these isolates and showed two possible sources of S. Gallinarum in the studied outbreaks.4. S. Gallinarum isolated from fowl typhoid outbreaks in the studied period were not caused by the use of the SG9R live vaccine. The source of strains sequenced was diverse.

Rapid detection and serovar identification of common Salmonella enterica serovars in Canada using a new pyrosequencing assay.

Serotyping of Salmonella enterica subsp. enterica is a critical step for foodborne salmonellosis investigation. To identify Salmonella enterica subsp. enterica serovars, we have developed a new assay based on a triplex polymerase chain reaction (PCR) with pyrosequencing for amplicon confirmation and phylogenetic discrimination of strains. The top 54 most prevalent serovars of S. enterica in Canada were examined with a total of 23 single-nucleotide polymorphisms (SNPs) and (or) single-nucleotide variations (SNVs) located on 3 genes (fliD, sopE2, and spaO). Seven of the most common serovars, Newport, Typhi, Javiana, Infantis, Thompson, Heidelberg, and Enteritidis, were successfully distinguished from the other serovars based on their unique SNP-SNV combinations. The remaining serovars, including Typhimurium, ssp I:4,[5],12:i:-, and Saintpaul, were further divided into 47 subgroups that demonstrate the relatedness to phylogenetic classifications of each serovar. This pyrosequencing assay is not only cost-effective, rapid, and user-friendly, but also provides phylogenetic information by analyzing 23 selected SNPs. With the added layer of confidence in the PCR results and the accuracy and speed of pyrosequencing, this novel method would benefit the food industry and provides a tool for rapid outbreak investigation through quick detection and identification of common S. enterica serovars in Canada.

Molecular Characterisation of Uropathogenic Escherichia coli Isolates at a Tertiary Care Hospital in South India

Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors (VFs) to break the inertia of the mucosal barrier of the urinary tract. The aim of the present study was undertaken to characterised the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients. A total of 156 non-repeated, clinically significant UPEC isolates were studied. Virulent genes were determined by two set of multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiograms and patient’s clinical outcomes were collected in a structured pro forma. Of the 156 patients infected by UPEC strains with significant bacterial counts the most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%). On analysis of the VF genes of the isolates, a majority of strains (140; 90%) were possessing the fimH gene followed by iutA (98; 63%), papC (76; 49%), cnf1 (46; 29.5%), hlyA (45; 29%) and neuC (8; 5%), respectively. On phylogenetic analysis, 27 (17%) isolates were belong to phylogroup A, 16 (10%) strains to Group B1, 59 (38%) were from Group B2 and 54 (35%) were from Group D. High prevalence of antibiotic resistance was observed among the isolates. The incidence of papC, cnf1 and hlyA was significantly higher (P < 0.05) among the isolates from relapse patients. Our findings indicate that virulent as well as commensal strains are capable of causing urinary tract infection. Virulence genes as well as patients-related factors are equally responsible for the development of infections and also that virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.

Phylogenetic Grouping and Phenotypic Detection of Extended-Spectrum β-Lactamases Among Escherichia coli From Calves and Dairy Cows in Khuzestan, Iran

Background: Food-producing animals are under suspicion for the reservoir and colonization of ESBL (extended-spectrum beta-lactamase)-producing bacteria especially Enterobacteriaceae and therefore infection of the humans with them. The increasing reports on the ESBLs presence in the pathogenic and commensal Escherichia coli isolates have been a concern worldwide. These strains can be attributed to one of the main phylogenetic groups and subgroups. Several studies have shown the relationship between the phylogeny and antimicrobial resistance of E. coli strains. Objectives: The aim of this study was to analyze the phylogenetic group of ESBL-producing E. coli and detect its phenotype using the multiplex polymerase chain reaction (PCR) and combined disk method. Materials and Methods: Two hundred five E. coli fecal isolates were obtained from 103 calves (90 healthy and 13 diarrheic) and 102 dairy cows (healthy) from 8 farms in Khuzestan province, Iran. The triplex PCR method was used to allocate the E. coli isolates based on the presence or absence of 3 genes (chuA, yjaA, and tspE4.C2) to yield 4 definite phylogenetic groups and 7 subgroups. Phenotypic ESBL-producing E. coli was determined using the double disk diffusion method according to the manufacturer’s instructions and Clinical &amp; Laboratory Standards Institute (CLSI) guidelines. Results: A total of 65.04% and 22.3% of isolates from calves and 70.5% and 20.5% of isolates from dairy cows belonged to phylogroups B1 and A, respectively. In addition, no isolate from the diarrhoeic calves was found to belong to group B2 and subgroups D2 and A0. A low prevalence (2/205 isolates, 0.97%) of ESBL-producing E. coli was found only in the samples of dairy cows which belonged to the phylogenetic group A and phylogenetic subgroup A1. There was no statistically significant relationship between the phylogenetic group and the production of ESBLs (P = 0.11). There was also no difference between the E. coli isolates from calves and dairy cows in the production of ESBLs (P = 0.5). Conclusion: There was no statistically significant relationship between the phylogenetic group and the production of ESBLs (P = 0.11). There was also no difference between E. coli isolates from calves and dairy cows in the production of ESBLs (P = 0.5). Based on these results, there is a low prevalence of ESBL-producing E. coli in the dairy farms of Khuzestan province. However, further large scale investigations are necessary to control the antibiotic resistance in the human and animal foodstuff.

Is diabetes mellitus an important risk factor for the antibiotic resistance in extraintestinal pathogenic Escherichia coli?

Escherichia coli is a major cause of extraintestinal infections in all age group. However, the infection becomes more severe when patients have some underlying condition such as Diabetes Mellitus. The aim of the study was to determine whether diabetic mellitus may act as an important risk factor for the E. coli to express drug resistance property. This descriptive study was carried out in a multi-specialty tertiary care hospital. One hundred and twenty-seven E. coli isolates from diabetic patients, and one hundred seventy-three isolates from nondiabetic patients were studied. Possession drug resistance genes were determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR. Antibiotic sensitivity testing was performed by Kirby-Bauer disk diffusion method. Among the study isolates from Diabetic patients maximum numbers were from phylogroup B2 (42.5%) and D (33%) similarly in case of nondiabetic patients B2 (29%) and D (38%) were the most common phylogroup. Presence of drug resistance genes among the diabetic and nondiabetic patient's isolates were as followed extended-spectrum beta-lactamase (70% and 70.5%) AmpC (9.5% and 14.5%) and NDM-1 ( 7% and 4.5%) and by disk diffusion methods susceptibility pattern were meropenem (94% and 94%), imipenem (92% and 92%), amikacin (76% and 74%), and ampicillin/sulbactam (68% and 69%), respectively. The proportion of diabetic patients strains with the drug resistance characteristics were not significantly different from that seen in nondiabetic patients strains, which indicating that in a predisposed host additional or subtraction bacterial aids for drug resistance property are not a necessity.

Multiplex PCR for detection of water-borne bacteria

Microbial water-borne diseases still affect developing countries and are major water quality concerns throughout the world. Routine culture-based methods of identifying bacterial pathogens in water sources are laborious and time-consuming. Recently, the use of molecular techniques such as the polymerase chain reaction (PCR) has provided rapid and highly promising detection methods. In this study, we developed two multiplex PCR assays for simultaneous detection of six water-borne bacteria. Two triplex PCR protocols were developed to detect six target genes. The first protocol targets uidA (Escherichia coli), int (Shigella spp.), and gyrB (Pseudomonas aeruginosa) genes, while invA (Salmonella spp.), ompW (Vibrio cholera), and lacZ (coliforms) were amplified by the second protocol. Specificity testing was carried out for 12 reference strains. Furthermore, the applicability of the multiplex PCR assays for detection of these bacteria was investigated for 52 surface water samples. The results indicated that all primer pairs showed specificities only for their corresponding target organisms. The detection sensitivity of both multiplex PCR assays was 3 × 102 − 3 × 103 colony forming units. The developed assays represent simple and efficient diagnostic procedures for co-detection of water-borne bacteria and have the potential to provide earlier warnings of possible public health threats and more accurate surveillance of these organisms.

Virulence factor profiles, phylogenetic background, and antimicrobial resistance pattern of lactose fermenting and nonlactose fermenting Escherichia coli from extraintestinal sources.

In recent years, nonlactose fermenting (NLF) Escherichia coli have been increasingly isolated in the microbiology laboratory, but their clinical significance has not yet been clearly elucidated. To characterize the lactose fermenting (LF) and NLF isolates on the basis of their virulence factors, phylogenetic background, and drug resistance property. This descriptive study was carried out in a multi-specialty tertiary care hospital. Three hundred nonrepeat E. coli isolates from inpatients were studied. Isolates were differentiated as LF and NLF on the basis of colony characteristics on MacConkey's agar. Possession of virulence and drug resistance genes was determined by multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiotic sensitivity testing was performed by disk diffusion method. Of 300 isolates 39 (13%) were NLF isolates. Maximum number of NLF isolates belonged to phylogroups B2 and D when compared with LF isolates. The incidence of iutA, hlyA, and neuC genes were significantly higher in NLF isolates. The presence of drug resistance genes such as AmpC gene, SHV, and CTXM were higher in LF isolates. LF isolates demonstrated a higher antimicrobial resistance and NLF isolates possessed higher virulence properties. The microbiology laboratory should report lactose fermentation profile as it may help the physician to initiate appropriate treatment.

Virulence Genes, Genetic Diversity, Antimicrobial Susceptibility and Phylogenetic Background of Escherichia coli Isolates

Background: The epidemiology of Uropathogenic Escherichia coli (UPEC) in urban and rural communities in Iran was never investigated prior to this study. Objectives: The aims of this study were to detect the frequency of virulence genes and determine the antimicrobial susceptibility and phylogenetic background of Escherichia coli isolates collected from urban and rural communities. Materials and Methods: A total of 100 E. coli isolates were collected from urine samples of patients with urinary tract infections (UTIs) residing in two different locations, and confirmed by current biochemical tests. The phylogenetic groups were determined by the triplexpolymerase chain reaction (PCR) method, and multiplex PCRs were used to detect eight Vf genes (fimH , iucD, irp2, hlyA, ompT, iha, iroN, and cnf1). The susceptibility profile of E. coli isolates was determined by the disk diffusion method. Results: Ninety-five percent of UPEC showed at least one of the virulence genes, the most prevalent being fimH (95%), followed by irp2 (89%), iucD (69%), ompT (67%), iroN (29%), and iha (29%). The various combinations of detected genes were designated as virulence patterns. Phylogenetic groups, B2 (55%) and D (22%), comprised the majority of isolated strains. Phenotypic tests showed that 92%, 74% and 71% of the isolates were resistant to ampicillin, ceftizoxime and cefixime, respectively. Conclusions: These findings indicate that the UPEC isolates had eight virulence factors with high frequencies. Moreover, these results suggest a direct connection between virulence factors, gene diversity, phylogenetic background, and antimicrobial resistance in UPEC isolates.

Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR

Background: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs.Objectives: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method.Results: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates.Conclusions: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.