Antibiotic Resistance and Plasmid Profile Analysis of Salmonella Enteritidis Isolated in Siberia and the Far East of Russia between 1990 and 2017

Salmonella is one of the major causes of foodborne disease outbreaks globally. Specifically, Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the major causes of zoonotic Salmonella infection in humans worldwide. In this study, we present data on antimicrobial resistance (AMR) and plasmid profiles of S. Enteritidis strains isolated from patients, food, and the environment in Siberia and the Far East of Russia obtained during Salmonella monitoring between 1990 and 2017. A total of 345 S. Enteritidis isolates were tested by the disk diffusion method with a set of 15 antibiotics using EUCAST breakpoints v. 10 and by plasmid profile analysis using the alkaline lysis method. The results have shown a substantial decrease in susceptibility to aminoglycosides and quinolones during the study period. No significant differences were found in the susceptibility of strains between regions as well as in the its correlation with different plasmid types of the pathogen. Several S. Enteritidis strains were found to be resistant to ampicillin, kanamycin, tetracycline, chloramphenicol, and cephalosporins. All tested S. Enteritidis strains were susceptible only to imipenem. In this study, we observed a relatively low level of AMR in S. Enteritidis strains isolated in Siberia and the Far East of Russia. Nevertheless, it is important to continue the molecular genetic monitoring and AMR surveillance of S. Enteritidis to track further increases in AMR using conventional phenotypic susceptibility testing and by introducing whole-genome sequencing to identify AMR mechanisms.

Amplification of cestode DNA from the peri-anal region of naturally infected foxes by PCR and LAMP: proof of concept for a potential sampling strategy for diagnosing human taeniosis

The diagnosis of human taeniosis can be achieved through coproscopy, ELISA or PCR. An important limitation of these methods is the high turnaround time for stool sample collection and preparation, indicating the need for a straightforward sampling strategy. Due to the high metabolic activity and reproductive potential of Taenia spp., we hypothesise that parasite DNA (cells and eggs) present in the peri-anal region of the host can be exploited as a target for molecular diagnosis. We evaluated the feasibility of recovering parasite DNA from the peri-anal area of foxes naturally infected with Taenia spp. Before necropsy, cotton swabs were rubbed at the peri-anal region of foxes. DNA was extracted using alkaline lysis coupled with a commercial DNA isolation kit (method A) or alkaline lysis alone (method B). DNA was used in the multiplex-PCR assay (previously described and called here swab-PCR) and a novel LAMP assay detecting Taenia spp. commonly found in foxes (swab-LAMP). The results of these assays from 105 foxes were compared with the presence of intestinal helminths determined at necropsy and by the sedimentation and counting technique (SCT). The sensitivity of swab-PCR for detecting Taenia (n = 68) was 89.8% (95% CI, 77.7–96.6) and 89.5% (66.9–98.7) using methods A and B, respectively. The sensitivity of the swab-LAMP assay was 83.7% (70.3–92.7) using method A and 89.5% (66.9–98.7) with method B. We postulate that peri-anal swab sampling followed by simplified DNA extraction and LAMP might be a suitable strategy for surveillance of human taeniosis in resource-limited settings in the future.

Antibiotic Resistance Pattern and Plasmid Profile of Bacteria Isolates from Household Water Distribution Tanks in Ado-Ekiti

Water is essential to life. The existence of all forms of life is dependent on an adequate water supply. The exigent need for water supply in homes prompted the construction of water sources and water storage devices in the homes. This however does not guarantee that the water is safe to drink. If the water is safe at the source, it may be contaminated during transportation storage and drawing at home. This study was carried out to determine the microbial counts, antibiotics susceptibility and plasmid profile of bacteria isolates from household water distribution tanks in the Ado-Ekiti metropolis. The total bacteria and coliform counts were determined using the pour plating technique. The antibiotic susceptibility pattern of the isolates was determined using the disc diffusion technique while the plasmid profile of the isolates was determined using the alkaline lysis method and agar gel electrophoresis. The mean total bacteria count of the water sample was 6.96 log10 CFU/ml, while the mean total of coliform count is 5.50 log10CFU/ml. The isolates with multiple antibiotics resistance belonged to five bacteria genera namely: Escherichia, Pseudomonas, Klebsiella, Enterobacter and Proteus. The plasmid analysis showed that four of the resistant strains had multiple plasmids, Enterobacter aerogens had 3 plasmids (1kb, 1.5kb and 2kb), Pseudomonas aeruginosa and Klebsiella aerogens had two plasmids (1kb, 1.5kb) respectively while Proteus vulgaris and Escherichia coli had no plasmid.

Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

PREVALENCE OF EXTENDED SPECTRUM BETA-LACTAMASES (ESBLS) PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE AMONG HOSPITALIZED PATIENTS FROM NIGERIA

The aim of this study was to determine the resistance patterns and ESBLs production among clinical isolates of Escherichia coli and Klebsiella pneumoniae in two government hospitals of Delta State, Nigeria. Urine, blood and wound samples were aseptically collected from hospitalized patients, bacteriologically processed and isolates identified using standard protocols. Antimicrobial susceptibility testing was determined by disc diffusion method. The plasmid DNA of Multidrug resistance (MDR) isolates were extracted by alkaline lysis method. Phenotypic ESBL production of the MDR isolates was done by Double Disc Synergy Test (DDST) while PCR was used to detect blaCTX-M, blaSHV and blaTEM among isolates. A total of 217 isolates were obtained, of which 161(74.2%) and 56(25.8%) were Escherichia coli and Klebsiella pneumoniae respectively. The antimicrobial resistance varied from one location to another. All isolates obtained from blood of general hospital Warri (GHW) were 100% resistant to amoxicillin clavulanic acid and the cephalosporins (ceftazidime, cefotaxime, and cefuroxime). Isolates from General hospital Agbor (GHA) showed high resistance of 75.0% to cefotaxime, 93.8% to each of ceftazidime and cefuroxime. Overall low resistance to nitrofurantoin was observed in E. coli isolates obtained from urine of GHW (27.5%) and GHA (20.8%). Out of 217 isolates, 75.1% (163/217) were MDR, of which 36.8% and 39.3% produced ESBL by DDST and PCR respectively. The most common ESBL gene was blaCTX-M expressed by 28(17.2%) of the isolates. The high prevalence of MDR and ESBL underscores the need for a continuous local monitoring of antibiotic resistance.

Antibiotic Sensitivity Pattern and Plasmid Profile of Bacteria Isolated from Diabetic Ulcers in Mbano Metropolis, Imo State, Southeastern Nigeria

The study was undertaken to evaluate the bacteriology and antibiogram of isolates from diabetic patients with chronic foot ulcers in Nigeria. A total of 150 pus samples were collected and processed according to standard aerobic and anaerobic microbiological methods. Antibiogram was done using Kirby-Bauer method. Biofilm tests, ESBL & AmpC production was conducted using Congo red agar, Double disc synergy test and Cefoxitin disc test respectively. Total number of isolates obtained was 210. The Plasmid profiles of some of the Multi-Drug Resistance (MDR) isolates were carried out using the alkaline lysis method for plasmid extraction and electrophoresis on agarose gel with standard markers. The most frequently isolated aerobic organism in the study was Escherichia coli (32.1%) while the least occurring was Enterobacter spp (1.57%). For the anaerobes, Peptostreptococcus spp (40%) was the highest isolated bacterium. Percentage of Extended Spectrum -lactamase ( ESBL) producers among E. coli isolates was 44%. Percentages of biofilm formation potential among the isolates were: E. coli (36.8%), S. aureus (23.1%) and Proteus vulgaris (4.2%). Escherichia coli and S. aureus showed considerable levels of resistance to some common antibiotics. No methicilin resistant S. aureus was encountered. AmpC producers encountered were Klebsiella pneumonia (10%) and E. coli (8.1%). Post-curring antibiogram tests revealed that nine isolates carried plasmids, suggesting that the mode of resistance may be plasmid mediated. Keywords: Diabetic ulcers, Bacteria, Antibiogram, Plasmid profile

Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification–lateral flow strip assay

Phytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

Antibiotic resistance and plasmid analysis of Enterobacteriaceae isolated from retail meat in Lagos Nigeria

Background The presence of antibiotic resistant microorganisms in food is of great concern globally. This research was carried out to detect and characterize plasmid carriage and profiles among members of Enterobacteriaceae from different meat types in Nigeria. Method From a total of 80 meat samples comprising of mutton,pork, beef and chicken, organisms belonging to the family Enterobacteriaceae wereisolated by standard procedures and identified by API 20E system. Antibiotics susceptibilities testing (AST) againstselected classes of antimicrobial agents and plasmid extraction was carried outby disc diffusion and alkaline lysis methods respectively. Results One-hundred and ten Enterobacteriaceae were isolated,species identification revealed isolates belonging to 7 genera comprising of Escherichia, Enterobacter, Klebsiella,Citrobacter, Proteus, Salmonella and Serratia. Overall resistance of theorganisms to amoxycillin/clavulanic acid was 91 (82.7%), streptomycin 85(75.7%) and perfloxacin 74 (67.2%) while ofloxacin had the highestsusceptibility rate (91.8%). Plasmids profiling revealed ranges of plasmids from1 to 3 copies with estimated sizes range of 700bp to 1.1kb among E. coli, K. pneumoniae, E. aerogenesand Proteus mirabilis. All theisolates with plasmids were multidrug resistant and were isolated from chicken excepta strain of E. coli from pork whichharboured a single plasmid copy suggesting these meat as reservoirs forantibiotic resistant bacteria. Conclusion Our findings revealed high level of meat contamination with antibioticresistant Enterobacteriaceae harbouring resistant plasmids. An integratedsurveillance system and safety practice must be ensured among the processorsand retailers.

Unlocking inaccessible historical genomes preserved in formalin

BackgroundMuseum specimens represent an unparalleled record of historical genomic data. However, the wide-spread practice of formalin preservation has thus far impeded genomic analysis of a large proportion of specimens. Limited DNA sequencing from formalin-preserved specimens has yielded low genomic coverage with unpredictable success. We set out to refine sample processing methods and to identify specimen characteristics predictive of sequencing success. With a set of taxonomically diverse specimens collected between 1936 and 2015 and ranging in preservation quality, we compared the efficacy of several end-to-end whole genome sequencing workflows alongside a k-mer-based trimming-free read alignment approach to maximize mapping of endogenous sequence.ResultsWe recovered complete mitochondrial genomes and up to 3X nuclear genome coverage from formalin-fixed tissues. Hot alkaline lysis coupled with phenol-chloroform extraction out- performed proteinase K digestion in recovering DNA, while library preparation method had little impact on sequencing success. The strongest predictor of DNA yield was overall specimen condition, which additively interacts with preservation conditions to accelerate DNA degradation.ConclusionsWe demonstrate a significant advance in capability beyond limited recovery of a small number of loci via PCR or target-capture sequencing. To facilitate strategic selection of suitable specimens for genomic sequencing, we present a decision-making framework that utilizes independent and non-destructive assessment criteria. Sequencing of formalin-fixed specimens will contribute to a greater understanding of temporal trends in genetic adaptation, including those associated with a changing climate. Our work enhances the value of museum collections worldwide by unlocking genomes of specimens that have been disregarded as a valid molecular resource.