Universal Polymerase Chain Reaction Research Articles

Development of a rapid, culture-free, universal microbial identification system using internal transcribed spacer targeting primers.

The indiscriminate administration of broad-spectrum antibiotics is a primary contributor to the increasing prevalence of antibiotic resistance. Unfortunately, culture, the gold standard for bacterial identification is a time intensive process. Due to this extended diagnostic period, broad-spectrum antibiotics are generally prescribed to prevent poor outcomes. To overcome the deficits of culture-based methods, we have developed a rapid universal bacterial identification system. The platform utilizes a unique universal polymerase chain reaction (PCR) primer set that targets the internal transcribed spacer (ITS) regions between conserved bacterial genes, creating a distinguishable amplicon signature for every bacterial species. Bioinformatic simulation demonstrates that at least 45 commonly isolated pathogenic species can be uniquely identified using this approach. We experimentally confirmed these predictions on a representative set of pathogenic bacterial species. We also show that the system can determine the corresponding concentration of each pathogen. Finally, we validated performance in clinical urinary tract infection samples.

The Utility and Limitations of Universal Polymerase Chain Reaction Screening for SARS-CoV-2 During Hospital Admission.

Universal polymerase chain reaction (PCR) screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on hospital admission is an effective approach to preventing coronavirus disease 2019 (COVID-19) outbreaks in medical facilities. However, false-positive test results due to a recent infection are a concern. We investigated the usefulness and limitations of universal PCR screening for SARS-CoV-2 on hospital admission in a real-world setting. We retrospectively analyzed 1320 attempted hospital admissions for 775 patients at the Department of Respiratory Medicine, Kyushu University Hospital, between January 1, 2022, and May 2, 2023. Thirty-nine out of 1201 PCR tests (3.2%) yielded a positive result, with 22 of these results being considered false positives on the basis of a recent infection. We found that 39% of cases showed a positive PCR result between 31 and 60 days after the onset of COVID-19, although the threshold cycle (Ct) for target 1 (ORF1ab gene) of the Cobas SARS-CoV-2 test (Roche Diagnostics, Basel, Switzerland) was >30 in most instances. Hospital admission based on the results of PCR screening for SARS-CoV-2 should take into account not only PCR positivity but also the Ct value and recent COVID-19 history.

Microbiología clásica y molecular en el diagnóstico de la endocarditis infecciosa

Introduction and objectivesIn recent years, molecular techniques for the aetiological diagnosis of infective endocarditis (IE) have attained relevance; however, they are not implemented in all laboratories. Our objective is to evaluate the usefulness of broad-spectrum polymerase chain reaction (PCR) technique followed by sequencing (universal PCR) on valve tissue as part of the IE diagnostic practice and to compare the results with classical methods. ResultsUniversal PCR showed a higher sensitivity than valve culture (80.8 vs 27.1%, P<.001) and allowed the identification of the causative agent or the confirmation of the aetiological diagnosis in case of discordance in 7.7% of patients. Regarding conventional diagnosis, blood culture stands out as the reference technique and Gram staining due to its high positive predictive value. The highest sensitivity and specificity was obtained considering classical and molecular microbiology together. The anatomopathological study presented a sensitivity of 83.9% and made it possible to assess the results of the microbiological diagnosis when there were discrepancies. ConclusionsUniversal PCR proved to be a valuable technique that completes the classical diagnosis. In an entity as complex as IE, it is necessary to use all the diagnostic tools at our disposal. All areas of laboratory diagnosis are complementary and not exclusive and should be assessed together.

Lumbar Osteomyelitis and Epidural Abscess Caused by Cardiobacterium hominis—First Reported Case in the Pediatric Population: The Role of Universal 16S rRNA Gene PCR and Sequencing

Abstract This report presents a case of a 14-year-old female patient diagnosed with lumbar (L2–L3) epidural abscess and vertebral osteomyelitis caused by Cardiobacterium hominis. The diagnosis was made using universal 16S rRNA polymerase chain reaction. To the best of our knowledge, this is the first reported case of C. hominis lumbar epidural abscess and vertebral osteomyelitis in pediatric population. This case serves to illustrate the potential value of polymerase chain reaction and DNA sequencing in diagnosing culture-negative vertebral osteomyelitis and the potential role of C. hominis in causing this infection.

Association of maternal SARS-CoV-2 infection at the time of admission for delivery with labor process and outcomes of vaginal birth: A cohort study.

This study aimed to investigate the impact of maternal SARS-CoV-2 infection at the time of admission for delivery on labor process and outcomes of vaginal birth. A cohort study was carried out at the Obstetrics Department of Anhui Provincial Hospital, China, where universal reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 infection was introduced for all women admitted for labor and delivery from December 1-31, 2022. Women were divided into positive and negative groups based on the test result. All women having a singleton vaginal birth were included in final analysis. The effect of SARS-CoV-2 positivity on labor process and outcomes of vaginal birth was estimated by regression analyses. Among a total of 360 women included, 87 had a positive SARS-CoV-2 test and 273 a negative test. Women in the positive group had an increased likelihood of having longer labor (median 9.3 vs 8.3 hours; sB [log-transformed] 0.19; 95% confidence interval [CI] 0.09-0.28), episiotomy (39.1% vs 23.8%; adjusted odds ratio [aOR] 2.31; 95% CI 1.27-4.21), grade III meconium-stained amniotic fluid (19.5% vs 7.0%; aOR 2.52; 95% CI 1.15-5.54) and postpartum hospital stay exceeding 37 hours (58.6% vs 46.5%; aOR 1.71; 95% CI 1.00-2.91). They had reduced rates exclusive breastfeeding (26.7% vs 39%; aOR 0.21; 95% CI 0.09-0.46) as well as mixed feeding (46.5% vs 52.2%; aOR 0.28; 95% CI 0.13-0.60) at 1 week postpartum. No significant differences were observed in other aspects of labor process and birth outcomes, including the uptake of labor analgesia, postpartum hemorrhage (>500 mL) or neonatal outcomes. A positive maternal SARS-CoV-2 test in labor among women having vaginal birth was associated with a slightly longer duration of labor, increased likelihood of episiotomy, increased incidence of grade III meconium-stained amniotic fluid, a longer postpartum hospital stay and a lower rate of breastfeeding 1 week postpartum. However, it did not have an adverse impact on other birth outcomes.

Universal PCR primers for identifying species of the genus Sanguisorba and other members of the family Rosaceae

Seven primers for the amplification of non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA fragments from various plant species of Rosaceae. Primers worked for species of the genus Sanguisorba and some species of the family Rosaceae. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology and evolution of plants.

Genotyping and Phylogenetic Analysis of Human Papillomaviruses in Formalin Fixed Paraffin Embedded Sections from Cervical Lesions in Duhok-Iraq.

Human papillomavirus (HPV) has been identified as an important causative factor in cervical cancer development. Cervical cancer is the fourth most prevalent malignant tumor among women globally. The purpose of this study was to investigate the prevalence and genotyping sequences of HPV in formalin-fixed paraffin-embedded (FFPE) cervical tissue using conventional polymerase chain reaction (PCR), and HPV-DNA sequencing. Retrospective cross-sectional study. Forty (FFPE) blocks with different cervical lesions were taken; patients' ages ranged from 24 to 65 years. Detection and sequencing of HPV DNA were done by conventional PCR (L1 gene), which was achieved by universal PCR primers (MY09/11 oligonucleotides). Then sequencing and phylogenetic tree was constructed. Nine samples were found positive and detected by conventional PCR, they were identified in CIN1 and SCC at 7.5% (n=3) for each, 5.0% (n=2) KA, and 2.5% (n=1) in CIN3 cases, after sequencing were submitted to GenBank and accession numbers were obtained. The phylogenetic tree was constructed and the aligned sequences showed high homology with the nucleotide sequence of the references from the Genbank database. HPV 11, 16, 18, 22, 33, 52, and 58 were found to have little nucleotide heterogeneity and thus no amino acid heterogeneity. Conclusion: Sequencing and phylogenetic analysis of circulating HPV types in Duhok provides very essential data about nucleotides and amino acid heterogeneity, to reveal genetic diversity with strains included in the vaccines that have not been introduced to Iraq yet.

From Field Tests to Molecular Tools-Evaluating Diagnostic Tests to Improve Rabies Surveillance in Namibia.

Rabies is endemic in Namibia and is present both in wildlife carnivores and domestic free-roaming dogs. The disease thus represents a challenge for public human and veterinary disease control. Namibia has implemented a national strategic plan to control rabies and the country's activities are supported by international organizations. To this end, rabies diagnosis at the Central Veterinary Laboratory (CVL) was improved in the frame of a World Organization for Animal Health (WOAH) laboratory twinning program: from practical sampling techniques and the use of lateral flow devices to a novel universal and discriminatory quantitative real-time Reverse transcription polymerase chain reaction (RT-qPCR), which easily identify dog-associated rabies viruses. The procedures applied and the results can be used as a template to improve rabies laboratory diagnosis.

536. Evaluation of the Clinical Utility of Universal Polymerase Chain Reaction Testing

Abstract Background Despite advances in microbiologic techniques, for patients with complex infections it often remains a challenge to identify the causative infectious pathogen. Traditional cultures (Cx) may fail to grow microorganisms due to inadequate sampling, prior antibiotic use, or the inherent insensitivity of culture methods for fastidious pathogens. Molecular tests allow for the detection of microbial nucleic acids directly from clinical specimens and do not require the presence of viable organisms for identification. Universal polymerase chain reaction testing (UPCR) is offered though the University of Washington Department of Laboratory Medicine and Pathology as a metagenomic approach using broad-range PCR primers followed by sequencing to hypothetically identify any pathogen present. The testing is composed of 3 separate tests for bacterial (BUPCR), fungal (FUPCR), and acid-fast (AFUPCR) organisms. The utility of UPCR has not been formally evaluated. Our objective is to describe the diagnostic utility of UPCR by comparing Cx and UPCR results, and their impact on management. Figure 1:Introduction - Types of Universal PCR Testing Methods We retrospectively collected data on UPCR and culture results and changes in antimicrobial therapy based on UPCR results for all patients with at least 1 UPCR test done during the 2-year study period. Results 367 UPCR tests were performed over 24 months on 155 patients. From 367 tests, 119 were FUPCR, 111 AFUPCR, and 137 BUPCR. 32/155 (20.6%) patients had positive UPCR. 25/32 were BUPCR and 7/32 were FUPCR. No AFUPCR was positive. In 8/155 (5.2%) patients management was changed based on UPCR results: Positive UPCR results directed treatment in 5 patients: 4 patients had positive UPCR and negative culture, and 1 had both UPCR and Cx positive but for different organisms. In all 5 therapy was changed in favor of UPCR result. All 5 tests were BUPCR.Negative UPCR led to antimicrobial discontinuation in 3 patients. 11/155 (7.1%) patients had negative UPCR and positive Cx, 10 of which were BUPCR and 1 AFUPCR. These results did not change management. Figure 2:Results – Type of Tissue and Test ResultsFigure 3:Results – Universal PCR Test Type and ResultsFigure 4:Results - Summary Conclusion Based on the real-world experience, UPCR results have limited impact on antimicrobial management in our institution. Further studies may try to identify clinical scenarios where UPCR may be of better clinical utility. Disclosures All Authors: No reported disclosures.

The new normal: a UK fertility clinic experience of universal RT-PCR SARS-CoV-2 testing

Following the temporary closure of fertility clinics in 2020 in many countries across the world, the SARS-CoV-2 pandemic has meant that the sector has had to rapidly adapt to novel ways of operating. The aim of this study was to investigate the efficacy and feasibility of universal real-time polymerase chain reaction testing at an IVF clinic within a UK tertiary referral centre. Between March and December 2020, we performed 2,401 SARS-CoV-2 RT-PCR tests on 1,215 individual patients, of which eight were positive (0.3%). Appropriate positive case identification allowed for delay in treatment initiation or cancellation as applicable. This has allowed our unit to continue to operate safely and efficiently.

Universal polymerase chain reaction screening for severe acute respiratory syndrome coronavirus 2 infections among patients who visited the Department of Otolaryngology-Head and Neck Surgery, Tohoku University Hospital

Universal polymerase chain reaction screening for severe acute respiratory syndrome coronavirus 2 infections among patients who visited the Department of Otolaryngology-Head and Neck Surgery, Tohoku University Hospital

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

The Ocular Microbiome Is Altered by Sampling Modality and Age.

BackgroundStudies of the ocular microbiome have used a variety of sampling techniques, but no study has directly compared different sampling methods applied to the same eyes to one another or to a reference standard of corneal epithelial biopsy. We addressed this lack by comparing the microbiome from three conjunctival swabs with those of corneal epithelial biopsy.MethodsTwelve eyes (11 patients) were swabbed by calcium alginate swab, cotton-tipped applicator, and Weck-Cel cellulose sponge before a corneal epithelial biopsy (48 samples). We then performed 16S rRNA gene sequencing and universal 16S rRNA gene real-time polymerase chain reaction. Negative/blank controls were used to eliminate contaminants. An analysis was performed to examine the concordance of the three swab types to corneal epithelial biopsy. The effect of patient age on the ocular microbiome as determined by epithelial biopsy was also examined.ResultsThe ocular microbiome from corneal epithelial biopsies consisted of 31 genera with a relative abundance of 1% or more, including Weisella, Corynebacterium, and Pseudomonas. Of the three swab types, Weck-Cel differed the most from corneal biopsies based on beta-diversity analysis. Cotton swabs were unable to capture the Bacteroides population seen on epithelial biopsy. Therefore, calcium alginate swabs seemed to be the closest to epithelial biopsies. Older patients (≥65 years old) had higher alpha diversity (P < 0.05) than younger patients. Differential abundance testing showed that there were 18 genera that were differentially abundant between the two age groups, including Streptococcus and eight members of the Proteobacteria phylum.ConclusionsWe demonstrate that ocular sampling method and patient age can greatly affect the outcome of sequencing-based analysis of the ocular microbiome.Translational RelevanceBy understanding the impact of different sampling methods on the results obtained from the ocular surface microbiome, future research on the topic will be more reproducible, leading to a better understanding of ocular surface microbiome in health and disease.

Granulomatous Tubulointerstitial Nephritis in a Kidney Transplant Recipient: Case Report and Review of the Literature

Granulomatous tubulointerstitial nephritis (GTIN) is a rare pathologic finding on kidney biopsy. GTIN can be associated with drugs, infection, systemic granulomatous disease, and tubulointerstitial nephritis with uveitis syndrome. We present a case of GTIN in a kidney transplant recipient (KTR) and a literature review of published cases of GTIN in KTRs. A 65-year-old man with a history of pulmonary and ocular tuberculosis (TB), who had undergone deceased donor kidney transplant 8 years prior, was admitted for acute kidney injury, hypercalcemia, and uveitis. His medications included rifabutin, isoniazid, and tacrolimus. Serum laboratory tests revealed creatinine of 2.65 mg/dL (baseline 1.1-1.5 mg/dL) and corrected calcium of 13.2 mg/dL. Hypercalcemia workup showed parathyroid hormone 7 pg/mL, 1,25(OH) vitamin D 54 pg/mL, parathyroid hormone-related peptide <2.0 pmol/L, and angiotensin-converting enzyme 47 U/L. Kidney biopsy showed GTIN with noncaseating granulomas. Universal polymerase chain reaction testing for acid-fast bacilli, fungus, and bacteria was negative. He was treated with prednisone, and his kidney function returned to baseline, and his hypercalcemia resolved. GTIN is a rare entity seen in less than 1% of transplanted kidney biopsies. The exactly etiology of this GTIN case remains unknown. TB could not be entirely ruled out, because the patient was receiving active anti-TB therapy. Our literature review showed infection to be the leading cause of GTIN in KTRs and that GTIN with concomitant uveitis remains exceedingly rare. Steroids may be useful in certain cases.

Universal Polymerase Chain Reaction Screening for Severe Acute Respiratory Syndrome Coronavirus 2 in Asymptomatic Patients Before Hospital Admission in Tokyo, Japan

ObjectivesUniversal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., the causative agent of coronavirus disease 2019 [COVID-19]) polymerase chain reaction (PCR) screening before admission has been adopted by several hospitals to prevent nosocomial SARS-CoV-2 transmission from asymptomatic and pre-symptomatic patients. However, screening usefulness remains unclear because it depends on the regional COVID-19 prevalence, and only a few large-scale studies have been reported. Here we describe the universal PCR screening performed in our hospital before admission of more than 12,000 patients and their attendants to evaluate the usefulness of the screening.MethodsWe retrospectively described the universal PCR screening results for asymptomatic patients and their attendants before planned admissions at a hospital in Tokyo, Japan, from August 3, 2020, through March 31, 2021. Nasopharyngeal swab samples were collected at an in-hospital PCR center.ResultsIn total, 12,133 persons (11,859 asymptomatic patients and 274 attendants) underwent PCR screening; nine (0.07%) tested positive for SARS-CoV-2 RNA.ConclusionsUniversal PCR screening may be useful for the advanced detection of SARS-CoV-2 infected patients with or without symptoms, which can be a potential source of nosocomial SARS-CoV-2 transmission.

An Optimization and Common Troubleshooting Solving in Polymerase Chain Reaction Technique

Many genetic researches now relies on the study of variants in genetic material through different, diverse of universal Polymerase Chain Reaction (PCR) methods. In that context, we are putting a highlighted on the most important fundamental aspects of PCR technology, which help researchers to clarify and reduce the majority problems and difficulties may face them in the laboratory work of any kind of PCR technology in general.

Graft-derived Cell-free DNA as a Noninvasive Biomarker of Cardiac Allograft Rejection: A Cohort Study on Clinical Validity and Confounding Factors.

Circulating graft-derived cell-free DNA (dd-cfDNA) is a new marker of cardiac allograft damage that is used for noninvasive rejection diagnostics. We performed dd-cfDNA (%) in heart transplant recipients during the first posttransplant year. In 87 patients, serial dd-cfDNA determination at predefined time-points was performed in 770 single samples. dd-cfDNA fraction (%) was measured using an established universal droplet digital polymerase chain reaction method, providing same-day turn-around. Rejection was diagnosed according to clinical parameters and biopsies. Median dd-cfDNA (%) was high (5.36%) immediately after reperfusion and decreased to a median (interquartile range) of 0.10% (0.05%-0.24%) in clinically stable patients by postoperative day 10. Compared to dd-cfDNA (%) samples in clinically stable patients, values were higher (P < 0.001) in biopsy-proven rejection ISHLT 1R (0.42% [0.15%-0.53%]) and 2R rejection (0.84% [0.39%-0.97%]). Moreover, dd-cfDNA (%) was already significantly increased 9-30 days before biopsy-proven rejection (0.36% [0.20%-0.61%]). An as yet unknown finding was a slightly, but significantly (P < 0.0001) higher dd-cfDNA (%) value in samples of stable patients with pericardial effusions (PEs) (n = 94; 0.18% [0.07%-0.30%]) compared to samples of non-PE patients (n = 132; 0.07% [0.04%-0.17%]). Using a cutoff of 0.35%, sensitivity and specificity of dd-cfDNA for cardiac rejection were 0.76 and 0.83 (area under the curve [AUC] ROC-curve: 0.81 [95% confidence interval, 0.73-0.89]). Omitting PE samples from the control group yielded an AUC of 0.86 [95% confidence interval, 0.76-0.95]. Samples drawn <12 hours after endomyocardial biopsy showed high (0.40% [0.15%-1.21%]) dd-cfDNA values, also in ISHLT0R (0.36% [0.10%-0.60%]). dd-cfDNA plasma values were significantly associated with cardiac rejection. However, PE or improper sampling (eg, shortly after biopsy) should be considered as confounders for rejection diagnoses using dd-cfDNA.

Morphometrical and Molecular Characterization of Oesophagostomum columbianum (Chabertiidae: Oesophagostominae) and Haemonchus contortus (Trichostrongylidae: Haemonchinae) Isolated from Goat (Capra hircus) in Sylhet, Bangladesh.

This study was aimed at describing two (2) intestinal nematodes from naturally infected native breed of goats (Capra hircus) in Bangladesh, identified as Oesophagostomum columbianum (Curtice, 1890) Stossich 1899 and Haemonchus contortus (Rudolphi, 1803) Cobb, 1898. The identification was made based on morphometric features and was confirmed by amplifying internal transcribed spacer (ITS) and cytochrome c oxidase (cox1) gene. Well-developed lateral alae, distinct cervical papillae anteriorly to esophageal expansion, and male spicule length (0.73-0.79 mm, n = 2) were characteristically observed in O. columbianum. At the same time, male spicule length (0.40-0.46 mm, n = 2) and position of female vulvar flap (4.30-4.54 mm from posterior end, n = 3) were observed in H. contortus. DNA sequence homology of the ITS and cox1 gene of both specimens revealed the same results, showing similarity to the GenBank sequences of O. columbianum (GenBank No. KC715827; JX188470) and H. contortus (GenBank No. KJ724377; HQ389229). Phylogenetic analysis computed by maximum livelihood (ML) from the ITS nucleotide sequences revealed that the O. columbianum and H. contortus isolates identified in this study were clustered in the same clade with isolates from China and Iran, respectively. This study, for the first time, illustrates the characteristics of O. columbianum and H. contortus in Bangladesh, combining both morphological and molecular data. The universal primer-based polymerase chain reaction (PCR) protocol could be an economical and efficient option for researchers from poor resource settings for precise identification of nematodes. The information generated in this study may contribute to formulating effective control strategies against these nematodes.

Performance and feasibility of universal PCR admission screening for SARS-CoV-2 in a German tertiary care hospital.

Anamnestic screening of symptoms and contact history is applied to identify coronavirus disease 2019 (COVID-19) patients on admission. However, asymptomatic and presymptomatic patients remain undetected although the viral load may be high. In this retrospective cohort study, all hospitalized patients who received polymerase chain reaction (PCR)admission testing from March 26th until May 24th, 2020 were included. Data on COVID-19-specific symptoms and contact history to COVID-19 cases were retrospectively extracted from patient files and from contact tracing notes.The compliance to the universal testing protocol was high with 90%. Out of 6940 tested patients, 27 new severe acute respiratory syndrome coronavirus-2infections (0.4%) were detected. Seven of those COVID-19 cases (26% of all new cases) were asymptomatic and had no positive contact history, but were identified through a positive PCR test. The number needed to identify an asymptomatic patient was 425 in the first wave of the epidemic, 1218 in the low incidence phase. The specificity of the method was above 99.9%.Universal PCR testing was highly accepted by staff as demonstrated by high compliance. The costs to detect one asymptomatic case in future studies need to be traded off against the costs and damage caused by potential outbreaks of COVID-19.

Characterization and Antimicrobial Resistance of Escherichia coli and Its Serotypes Isolated from Poultry Feed in Relation to Seasons in Karachi, Pakistan

Background: The increasing prevalence of Escherichia coli as a foodborne pathogen in poultry poses a high risk to food safety. The resistant strains of E. coli may contribute resistant genes to human endogenous flora, causing fatal diseases. Moreover, these pathogens are serious threats to poultry farming. Objectives: The current study aimed to investigate the effects of seasonal variations on the bacterial load of E. coli and to evaluate its antibiogram profile. Methods: All feed samples were evaluated for the identification of E. coli and its serotypes, using conventional culture methods and biochemical characterization. Positive samples were confirmed by polymerase chain reaction (PCR) assay. The bacterial load of E. coli was estimated by measuring the total viable count, and the antibiogram data were calculated using two methods, that is, disc diffusion method and minimum inhibitory concentration (MIC) measurement. Results: Of 204 feed samples investigated, 38 isolates were positive for E. coli. All positive samples were also confirmed via universal and species-specific PCR assays, and 8/38 were documented as E. coli 0157:H7 strains. The bacterial load of E. coli was also determined by measuring the total viable count, and the results revealed the highest ratio (6.44×108 CFU/g) from June to August and the lowest ratio (2.06×108 CFU/g) from December to February. The multidrug resistance of E. coli O157:H7 was validated by antimicrobial susceptibility tests since all isolates showed high resistance to chloramphenicol, penicillin derivatives, fluoroquinolones, and oxytetracycline, respectively, and were only susceptible to aminoglycosides. Conclusions: Considering the high bacterial load of E. coli from June to August, the poultry industry needs to establish appropriate and effective hygienic and storage policies, especially during these alarming months. Moreover, surfacing and propagation of resistant strains of these pathogens may obscure future assessments for treatment purposes.