Prevalence and Characterization of Quinolone Resistance in Campylobacter spp. Isolates in Chicken Livers from Retail Stores in Georgia, USA

Campylobacter is the leading bacterial pathogen that causes human foodborne illnesses worldwide and outbreaks have been associated with consumption of under-cooked chicken livers. The objectives of this study were to compare two PCR assays for speciation of 250 Campylobacter isolates, to assess antibiotic resistance of the isolates, and to analyze genetic diversity of the quinolone resistance determining regions (QRDR) of the isolates. Speciation was performed in a double-blind manner, and results showed that 181 (72%) isolates were identified as Campylobacter jejuni and 69 (28%) isolates were identified as Campylobacter coli by both PCR assays. A total of 93 (37.2%) isolates were determined to be resistant to at least one antibiotic. Among 88 C. jejuni isolates, 33 isolates (18%) were resistant to nalidixic acid (NAL) and ciprofloxacin (CIP), followed by 25 (14%) isolates resistant to tetracycline (TET) and 18 (10%) isolates resistant to NAL and TET. Two isolates were resistant to four antibiotics tested. One isolate was resistant to five antibiotics tested. For C. coli, two isolates were resistant to TET, and two were resistant to NAL, CIP and TET. The amino acid sequences of the QRDR regions among the isolates revealed eight point mutations and could be classified into 12 groups. Thirty-eight C. jejuni isolates resistant to NAL and CIP had a point mutation at residue 86 (Thr to Ile substitution). However, six isolates without the substitution at the same position were resistant to NAL and/or CIP. In addition, 10 isolates with a point mutation at residue 86 were susceptible to NAL and CIP. This observation suggests that besides the substitution at 86, other mechanisms may confer resistance to quinolones. Further studies are needed to elucidate mechanisms for quinolone resistance in Campylobacter. Based on our results, the Campylobacter spp. isolated from chicken livers were found to be resistant to the quinolones and other classes of antibiotics.

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Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

10.1101/194936 ◽

2017 ◽

Author(s):

Sergey V. Prykhozhij ◽

Charlotte Fuller ◽

Shelby L. Steele ◽

Chansey J. Veinotte ◽

Babak Razaghi ◽

...

Keyword(s):

Point Mutation ◽

Point Mutations ◽

Mutation Site ◽

Transmission Rates ◽

Specific Pcr ◽

Allele Specific ◽

Pcr Assays ◽

Combination Of Methods ◽

Mutation Strategies ◽

Allele Specific Pcr

AbstractSingle-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

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Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v84i1.1411 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 13

Author(s):

Samantha Reddy ◽

Oliver T. Zishiri

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Antibiotic Resistance Genes ◽

Quinolone Resistance ◽

Campylobacter Coli ◽

Antimicrobial Resistance Genes ◽

Campylobacter Spp ◽

Clinical Cases

Campylobacter spp. are common pathogenic bacteria in both veterinary and human medicine. Infections caused by Campylobacter spp. are usually treated using antibiotics. However, the injudicious use of antibiotics has been proven to spearhead the emergence of antibiotic resistance. The purpose of this study was to detect the prevalence of antibiotic resistance genes in Campylobacter spp. isolated from chickens and human clinical cases in South Africa. One hundred and sixty one isolates of Campylobacter jejuni and Campylobacter coli were collected from chickens and human clinical cases and then screened for the presence of antimicrobial resistance genes. We observed a wide distribution of the tetO gene, which confers resistance to tetracycline. The gyrA genes that are responsible quinolone resistance were also detected. Finally, our study also detected the presence of the blaOXA-61, which is associated with ampicillin resistance. There was a higher (p < 0.05) prevalence of the studied antimicrobial resistance genes in chicken faeces compared with human clinical isolates. The tetO gene was the most prevalent gene detected, which was isolated at 64% and 68% from human and chicken isolates, respectively. The presence of gyrA genes was significantly (p < 0.05) associated with quinolone resistance. In conclusion, this study demonstrated the presence of gyrA (235 bp), gyrA (270 bp), blaOXA-61 and tetO antimicrobial resistance genes in C. jejuni and C. coli isolated from chickens and human clinical cases. This indicates that Campylobacter spp. have the potential of resistance to a number of antibiotic classes.

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Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens

Antibiotics ◽

10.3390/antibiotics10050594 ◽

2021 ◽

Vol 10 (5) ◽

pp. 594

Author(s):

Sydney E. Schnur ◽

Raghavendra G. Amachawadi ◽

Giovanna Baca ◽

Sarah Sexton-Bowser ◽

Davina H. Rhodes ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Phenolic Compounds ◽

Enterococcus Faecalis ◽

Campylobacter Jejuni ◽

Bacterial Pathogens ◽

Klebsiella Oxytoca ◽

Bovine Mastitis ◽

Campylobacter Coli ◽

Foodborne Illnesses ◽

Phenolic Extract

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study’s objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included Escherichia coli, Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus, and Enterococcus faecalis. Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18–24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, and Campylobacter coli.

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Lorlatinib Induces Durable Disease Stabilization in a Pancreatic Cancer Patient with a ROS1 p.L1950F Mutation: Case Report

Oncology Research and Treatment ◽

10.1159/000517616 ◽

2021 ◽

pp. 1-7

Author(s):

Janna-Lisa Velthaus ◽

Peter Iglauer ◽

Ronald Simon ◽

Carsten Bokemeyer ◽

Peter Bannas ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Patient ◽

Point Mutation ◽

Kinase Inhibitors ◽

Point Mutations ◽

Progression Free Survival ◽

Pancreatic Cancer Patient ◽

Targeted Treatment ◽

Precision Oncology ◽

Disease Stabilization

Introduction: The prognosis of pancreatic cancer has improved only modestly in recent years. This is partly due to the lack of development in precision oncology including immune oncology in this entity. Rearrangements of the proto-oncogene tyrosine protein kinase ROS1 gene represent driver alterations found especially in lung cancer. Tyrosine kinase inhibitors (TKI) with activity against ROS1 including lorlatinib substantially improved the outcome of this patient population. Anecdotal evidence reports treatment of pancreatic cancer harboring ROS1 fusions with ROS1 TKI, but data concerning treatment of patients with ROS1 point mutations are lacking. Case Presentation: This case describes a pancreatic cancer patient harboring a ROS1 point mutation that occurred without an underlying ROS1 rearrangement and thus not in the resistance situation. The heavily pretreated patient showed a strong decrease of the tumor biomarkers (CA19-9 and CEA) and radiologically a durable stable disease to the targeted treatment with lorlatinib, thereby achieving a progression-free survival of 12 months. Conclusion: Our data are the first to show a clinical benefit from targeted treatment with ROS1 TKI in a cancer patient with a thus far undescribed ROS1 point mutation without a concomitant ROS1 rearrangement. Furthermore, they indicate that ROS1 could be an oncogenic driver in pancreatic cancer. This subgroup could be eligible for targeted treatments, which may contribute to the urgently needed improvement in patient outcome.

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Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00439-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Saeed Sharifi ◽

Bita Bakhshi ◽

Shahin Najar-peerayeh

Keyword(s):

Gene Expression ◽

Point Mutation ◽

Campylobacter Jejuni ◽

Significant Contribution ◽

Efflux Pump ◽

Campylobacter Coli ◽

Rapd Pcr ◽

Resistant Strains ◽

High Level ◽

Level Resistance

Abstract Background Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. Methods A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. Results Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. Conclusion Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

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Altered toxicity of the prion protein peptide PrP106–126 carrying the Ala117→Val mutation

Biochemical Journal ◽

10.1042/bj3460785 ◽

2000 ◽

Vol 346 (3) ◽

pp. 785-791 ◽

Cited By ~ 2

Author(s):

David R. BROWN

Keyword(s):

Point Mutation ◽

Prion Protein ◽

Prion Diseases ◽

Point Mutations ◽

Human Sequence ◽

Gene Coding ◽

Normal Human ◽

Microtubule Formation ◽

Β Sheet ◽

Prion Protein Peptide

The inherited prion diseases such as Gerstmann-Sträussler-Scheinker syndrome (GSS) are linked to point mutations in the gene coding for the cellular isoform of the prion protein (PrPC). One particular point mutation A117V (Ala117 → Val) is linked to a variable pathology that usually includes deposition of neurofibrillary tangles. A prion protein peptide carrying this point mutation [PrP106-126(117V)] was generated and compared with a peptide based on the normal human sequence [PrP106-126(117A)]. The inclusion of this point mutation increased the toxicity of PrP106-126 which could be linked to an increased β-sheet content. An assay of microtubule formation in the presence of tau indicated that PrP106-126 decreased the rate of microtubule formation that could be related to the displacement of tau. PrP106-126 carrying the 117 mutation was more efficient at inhibiting microtubule formation. These results suggest a possible mechanism of toxicity for protein carrying this mutation via destabilization of the cytoskeleton and deposition of tau in filaments, as observed in GSS.

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Prevalence and Antimicrobial Resistance of Campylobacter spp. Isolated from Poultry Carcasses in Poland

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-035 ◽

2013 ◽

Vol 76 (8) ◽

pp. 1451-1455 ◽

Cited By ~ 23

Author(s):

KINGA WIECZOREK ◽

IWONA KANIA ◽

JACEK OSEK

Keyword(s):

Campylobacter Jejuni ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Genetic Determinants ◽

Gyra Gene ◽

23S Rrna Gene ◽

Pcr Method ◽

Almost All ◽

Campylobacter Spp

The purpose of the present study was to determine the prevalence of Campylobacter in poultry carcasses at slaughter in Poland. For the isolated strains, resistance to selected antibiotics and the associated genetic determinants were identified. A total of 498 Campylobacter isolates were obtained from 802 poultry samples during the 2-year study period. Strains were identified to species with the PCR method; 53.6% of the strains were Campylobacter jejuni and 46.4% were Campylobacter coli. A high percentage of the tested Campylobacter strains were resistant to ciprofloxacin and nalidixic acid (74.1 and 73.5%, respectively) followed by tetracycline (47.4%) and streptomycin (20.5%). Only one C. jejuni and two C. coli isolates were resistant to gentamicin. Seventy-nine (15.9%) of the 498 strains were resistant to three or more classes of antibiotics examined. Higher levels of resistance, irrespective of the antimicrobial agent tested, were found within the C. coli group. Almost all strains resistant to quinolones (99.5%) and to tetracycline (99.6%) carried the Thr-86-to-Ile mutation in the gyrA gene and possessed the tet(O) marker, respectively. All isolates resistant to erythromycin had the A2075G mutation in the 23S rRNA gene. These results reveal that poultry carcasses in Poland are a reservoir of potentially pathogenic and antimicrobial-resistant Campylobacter strains for humans, which may pose a public health risk.

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Mechanisms of quinolone resistance and implications for human and animal health

Veterinarski glasnik ◽

10.2298/vetgl1004277v ◽

2010 ◽

Vol 64 (3-4) ◽

pp. 277-285

Author(s):

Maja Velhner ◽

Gordana Kozoderovic ◽

Zora Jelesic ◽

Igor Stojanov ◽

Radomir Ratajac ◽

...

Keyword(s):

Veterinary Medicine ◽

Antimicrobial Resistance ◽

Protein Interactions ◽

Bacterial Infections ◽

Animal Health ◽

Point Mutations ◽

Inhibitory Effect ◽

Quinolone Resistance ◽

Topoisomerase Iv ◽

Target Enzyme

Quinolone antibiotics have been widely used in human and veterinary medicine. This has caused the development of resistance and difficulties in the treatment of complicated bacterial infections in humans. The resistance to quinolones develops due to chromosome mutations and it can also be transferred by plasmids. The target enzyme for quinolones in Gram-negative bacteria is Gyrasa A, while the target enzyme in Grampositive bacteria is mostly topoisomerase IV. Gyrase A consists of two subunits encoded by genes gyrA and gyrB. The function of the enzyme is to introduce negative super coiling in DNA and therefore is essential for the replication of bacteria. Quinolone resistance develops if point mutations at 83 and/or 87 codon are introduced on gyrA. Establishing a minimal inhibitory concentration (MIC) to this group of antimicrobials will reveal possible mutations. Recently it was discovered that quinolone resistance is transmittable by plasmid termed PMQR (plasmid mediated quinolone resistance). The target gene marked qnr encodes a pentapeptide repeat family protein. Pentapeptide repeats form sheets, involved in protein-protein interactions. Qnr protein binds to GyrA protecting the enzyme from the inhibitory effect of ciprofloxacin. The distribution of qnr related resistance is higher in humans than in animals. In poultry, however, this type of resistance is present more than in other animals. Plasmid mediated resistance contributes to the faster spread of quinolone resistance. Proper food handling will significantly contribute to decreasing the risk from infection to which people are exposed. In medical and veterinary laboratories antimicrobial resistance monitoring in clinical and environmental isolates is advised. Since correlation between antibiotics application and antimicrobial resistance is often suggested, antimicrobial use must be under strict control of the authorities both in human and in veterinary medicine. .

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Presence of the knockdown resistance (kdr) mutations in the head lice (Pediculus humanus capitis) collected from primary school children of Thailand

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008955 ◽

2020 ◽

Vol 14 (12) ◽

pp. e0008955

Author(s):

Narisa Brownell ◽

Sakone Sunantaraporn ◽

Kobpat Phadungsaksawasdi ◽

Nirin Seatamanoch ◽

Switt Kongdachalert ◽

...

Keyword(s):

Head Louse ◽

Point Mutations ◽

Human Head ◽

Amino Acid Sequences ◽

Primary School Children ◽

Head Lice ◽

Knockdown Resistance ◽

Pediculosis Capitis ◽

Α Subunit ◽

Blood Sucking

Human head lice are blood-sucking insects causing an infestation in humans called pediculosis capitis. The infestation is more prevalent in the school-aged population. Scalp itching, a common presenting symptom, results in scratching and sleep disturbance. The condition can lead to social stigmatization which can lead to loss of self-esteem. Currently, the mainstay of treatment for pediculosis is chemical insecticides such as permethrin. The extended use of permethrin worldwide leads to growing pediculicide resistance. The aim of this study is to demonstrate the presence of the knockdown resistance (kdr) mutation in head lice populations from six different localities of Thailand. A total of 260 head lice samples in this study were collected from 15 provinces in the 6 regions of Thailand. Polymerase chain reaction (PCR) was used to amplify the α subunit of voltage-sensitive sodium channel (VSSC) gene, kdr mutation (C→T substitution). Restriction fragment length polymorphism (RFLP) patterns and sequencing were used to identify the kdr T917I mutation and demonstrated three genotypic forms including homozygous susceptible (SS), heterozygous genotype (RS), and homozygous resistant (RR). Of 260 samples from this study, 156 (60.00%) were SS, 58 (22.31%) were RS, and 46 (17.69%) were RR. The overall frequency of the kdr T917I mutation was 0.31. Genotypes frequencies determination using the exact test of Hardy-Weinberg equilibrium found that northern, central, northeastern, southern, and western region of Thailand differed from expectation. The five aforementioned localities had positive inbreeding coefficient value (Fis > 0) which indicated an excess of homozygotes. The nucleotide and amino acid sequences of RS and RR showed T917I and L920F point mutations. In conclusion, this is the first study detecting permethrin resistance among human head lice from Thailand. PCR-RFLP is an easy technique to demonstrate the kdr mutation in head louse. The data obtained from this study would increase awareness of increasing of the kdr mutation in head louse in Thailand.

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Mutational Analysis of Quinolone-Resistant Determining Region gyrA and parC Genes in Quinolone-Resistant ESBL-Producing E. Coli

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v20i3.1825 ◽

2021 ◽

Vol 20 (3) ◽

Author(s):

Hairul Aini Hamzah ◽

Rahmatullah Sirat ◽

Mohammed Imad A. Mustafa Mahmud ◽

Roesnita Baharudin

Keyword(s):

Mutational Analysis ◽

Single Point ◽

Point Mutations ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Resistant Bacteria ◽

Single Point Mutation ◽

Phenotypic Resistance ◽

Drug Effectiveness ◽

E Coli

Introduction: Co-resistance to quinolones among extended spectrum β[1]lactamase (ESBL)-producing E. coli commonly occurs in clinical settings. Quinolones act on DNA gyrase and DNA topoisomerase enzymes, which are coded by gyrA and parC genes, thus any mutation to the genes may affect the drug effectiveness. The objective of the study was to characterize gyrA and parC genes in quinolone-resistant E. coli isolates and correlated the mutations with their phenotypic resistance. Materials and Methods: Thirty-two quinolone-resistant (QR) and six quinolone-sensitive (QS) ESBL-E. coli isolates were identified by antibiotic susceptibility and minimum inhibitory concentration tests. Bioinformatics analysis were conducted to study any mutations occurred in the genes and generate their codon compositions. Results: All the QR ESBL-E. coli isolates were identified as multidrug-resistant bacteria. A single point mutation in the quinolone resistance-determining region (QRDR) of gyrA, at codon 83, caused the substitution amino acid Ser83Leu. It is associated with a high level of resistance to nalidixic acid. However, double mutations Ser83Leu and Asp87Asn in the same region were significantly linked to higher levels of resistance to ciprofloxacin. Cumulative point mutations in gyrA and/or in parC were also correlated significantly (p<0.05) to increased resistance to ciprofloxacin. Conclusion: Together, the findings showed that the mutations in gyrA and parC genes handled the institution of intrinsic quinolone resistance in the ESBL-E. coli isolates. Thus, vigilant monitoring for emergence of new mutation in resistance genes may give an insight into dissemination of QR ESBL-E. coli in a particular region.

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