Method for Determining the Profile of Antibiotic Resistance Genes in the Vibrio cholera Strains by RT-PCR

The aim of the work was to design and carry out experimental studies of a set of reagents to identify the spectrum of genes that determine the resistance of the Vibrio cholerae strains to antibacterial drugs.Materials and methods. V. cholerae strains isolated from humans and environmental objects during epidemiological complications and the cholera-free period were included in the study. Sensitivity to antimicrobial drugs was evaluated by the disk diffusion method. Whole genome sequencing was performed on an Illumina MiSeq. The profile of resistance genes was determined based on a comparison with the ResFinder database. The temperature regime, the composition of the reaction mixtures, and the reaction parameters were optimized; the specificity, sensitivity and reproducibility of the constructed prototype test system were measured.Results and discussion. The spectrum of antibiotic resistance and the profile of resistance genes were determined for the studied strains. To develop multiplex PCR, we selected the most common in the V. cholerae populations genes, which are responsible for resistance to tetracycline (tetA), streptomycin (strA), florfenicol/ chloramphenicol (floR) and trimethoprim/sulfamethoxazole (two variants of the dihydrofolate reductase gene: dfrA1 and dhfR), as well as SXT element integrase gene (int). In the reaction, markers were specifically detected in accordance with the genomic resistance profile, which correlates with the phenotypic manifestation of resistance determined by the disco-diffusion method. The sensitivity of the developed panel of primers and probes for V. cholerae strains was 103 –104 CFU/ml. Therefore, taking into account the specificity, rapidity and simplicity of the reaction, the developed system of primers and probes can be successfully applied for a preliminary assessment of the resistance of the V. cholerae strains to antimicrobial agents.

Download Full-text

Prevalence of class 1 and 2 integrons among the multidrug resistant uropathogenic strains of Escherichia coli

Asian Biomedicine ◽

10.5372/1905-7415.0901.367 ◽

2017 ◽

Vol 9 (1) ◽

pp. 49-54 ◽

Cited By ~ 1

Author(s):

Haddadi Azam ◽

Somayeh Mikaili Ghezeljeh ◽

Shavandi Mahmoud

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Diffusion Method ◽

Disk Diffusion Method ◽

Integrase Gene ◽

Class 1 ◽

Tract Infections

Abstract Background Multidrug resistance is a serious problem in the treatment of urinary tract infections. Horizontal gene transfer, directed by strong selective pressure of antibiotics, has resulted in the widespread distribution of multiple antibiotic resistance genes. The dissemination of resistance genes is enhanced when they are trapped in integrons. Objectives To determine the prevalence of integrons among multidrug resistant Escherichia coli strains collected from regional hospitals and private clinical laboratories in Alborz province. Methods The susceptibility of 111 clinical Escherichia coli isolates was tested using a Kirby–Bauer disk diffusion method for common antibiotics. Isolates were screened for the production of extended spectrum β-lactamases (ESBLs) using a double disk synergy test. The existence of integrons was confirmed by amplification of the integrase gene and their class determined via analysis of PCR products by PCR-RFLP. Results Isolates showed the highest resistance to amoxicillin. Nitrofurantoin, amikacin, and ceftizoxime were the most effective antibiotics in vitro. Eighty-eight isolates of 111 (79%) were resistant to more than three unrelated drugs. We found 30% of the multidrug resistant isolates harbor integrons. Class 1 and 2 integrons were detected in 25 and 1 isolates, respectively. ESBL screening of strains showed 45 isolates (40%) were positive; 22% of the ESBL-positive isolates carried class 1 integrons and the frequency of MDR in ESBLpositive isolates was 93%. Conclusion The existence of integrons in only 29.5% of multidrug resistant isolates showed that besides integrons, antibiotic resistance genes were probably carried on other transferable elements lacking integrons, such as transposons or plasmids.

Download Full-text

Preliminary Results on the Prevalence of Salmonella Spp. in Marine Animals Stranded in Sicilian Coasts: Antibiotic Susceptibility Profile and ARGs Detection in the Isolated Strains

Pathogens ◽

10.3390/pathogens10080930 ◽

2021 ◽

Vol 10 (8) ◽

pp. 930

Author(s):

Delia Gambino ◽

Sonia Sciortino ◽

Sergio Migliore ◽

Lucia Galuppo ◽

Roberto Puleio ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Susceptibility ◽

Antibiotic Resistance Genes ◽

Diffusion Method ◽

Salmonella Spp ◽

Marine Animals ◽

Disk Diffusion Method ◽

Phenotypic Resistance ◽

Low Prevalence

The presence of Salmonella spp. in marine animals is a consequence of contamination from terrestrial sources (human activities and animals). Bacteria present in marine environments, including Salmonella spp., can be antibiotic resistant or harbor resistance genes. In this study, Salmonella spp. detection was performed on 176 marine animals stranded in the Sicilian coasts (south Italy). Antibiotic susceptibility, by disk diffusion method and MIC determination, and antibiotic resistance genes, by molecular methods (PCR) of the Salmonella spp. strains, were evaluated. We isolated Salmonella spp. in three animals, though no pathological signs were detected. Our results showed a low prevalence of Salmonella spp. (1.7%) and a low incidence of phenotypic resistance in three Salmonella spp. strains isolated. Indeed, of the three strains, only Salmonella subsp. enterica serovar Typhimurium from S. coeruleoalba and M. mobular showed phenotypic resistance: the first to ampicillin, tetracycline, and sulphamethoxazole, while the latter only to sulphamethoxazole. However, all strains harbored resistance genes (blaTEM, blaOXA, tet(A), tet(D), tet(E), sulI, and sulII). Although the low prevalence of Salmonella spp. found in this study does not represent a relevant health issue, our data contribute to the collection of information on the spread of ARGs, elements involved in antibiotic resistance, now considered a zoonosis in a One Health approach.

Download Full-text

Characterization of Vibrio parahaemolyticus strains isolated in Chile in 2005 and in 2007

The Journal of Infection in Developing Countries ◽

10.3855/jidc.1228 ◽

2010 ◽

Vol 5 (07) ◽

pp. 502-510 ◽

Cited By ~ 12

Author(s):

Priscila Dauros ◽

Helia Bello ◽

Mariana Domínguez ◽

Juan C. Hormazábal ◽

Gerardo González

Keyword(s):

Antibiotic Resistance ◽

Antibiotic Resistance Genes ◽

Diffusion Method ◽

Clinical Samples ◽

Disk Diffusion Method ◽

Integrase Gene ◽

Class 1 ◽

Single Clone ◽

Almost All ◽

Pfge Profiles

Introduction: Vibrio (V.) parahaemolyticus has endemically established in Chilean sea shores, causing outbreaks every year, with an important number of cases. In order to know the genetic relationship, genotype dominance and antibiotic resistance of isolates obtained from two outbreaks, this study characterized 110 strains isolated from environmental and clinical samples in years 2005 and 2007 in Chile. Methodology: Genotyping was performed by determination of PFGE profiles, and pandemic group and integrons were screened by PCR. Antimicrobial susceptibility was studied by the disk diffusion method. Results: High antibiotic susceptibility frequency was found, mainly among 2007 isolates, except to ampicillin, cephalothin, cefoxitin, cefpodoxime, amikacin, streptomycin and kanamycin. Strains belonging to the pandemic group in clinical isolates account for 88% in 2005, decreasing to 66% in 2007 and among environmental isolates were detected in 20% of the strains from 2005, rising to 36% in 2007. In 2005, nine different PFGE profiles were identified, with 78% of the strains corresponding to a single clone. In 2007, sixteen different PFGE profiles were detected, with 61% of the strains included into a sole clone. The same clone was prevalent in both years. None of class 1, 2, 3 and SXT integrases genes was detected; however, the superintegron integrase gene (intIA) was present in almost all strains. Conclusions: These results suggest the persistence and dominance of a unique PFGE clone of V. parahaemolyticus during 2005 and 2007, and the absence of genetic elements that capture antibiotic resistance genes described in other species of Vibrio.

Download Full-text

Antimicrobial Susceptibility, Serotyping, and Molecular Characterization of Antibiotic Resistance Genes in Listeria monocytogenes Isolated from Pregnant Women with a History of Abortion

Iranian Journal of Public Health ◽

10.18502/ijph.v50i1.5084 ◽

2021 ◽

Author(s):

Siamak HEIDARZADEH ◽

Mohammad Reza POURMAND ◽

Saeedeh HASANVAND ◽

Reyhaneh PIRJANI ◽

Davoud AFSHAR ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Pregnant Women ◽

Resistance Genes ◽

Antimicrobial Agents ◽

Tertiary Care ◽

Antibiotic Resistance Genes ◽

Diffusion Method ◽

Care Hospital ◽

Disk Diffusion Method ◽

History Of

Background: Listeria monocytogenes show high mortality among pregnant women and newborns. This study aimed to detect L. monocytogenes in pregnant women with a history of abortion and assess the serotypes, antibiotic susceptibility patterns, and its resistance genes. Methods: Overall, 400 vaginal swabs were taken from pregnant women with a history of abortion in the past few years in a tertiary care hospital in Tehran, Iran, during 2015-2018. Antibiotics susceptibility to a panel of 10 antibiotics was determined using the standard disk diffusion method and the isolates serotyped by the agglutination method. The antimicrobial-resistant isolates were also screened for the presence of tetM, ermB and dfrD genes by PCR. Results: Overall, 22 L. monocytogenes isolates were identified. High rates of resistance were observed for trimethoprim (50%; n=11), sulphamethoxazole (50%; n=11), tetracycline (45.45%; n=10) and gentamicin (36.36%; n=8). From 22 L. monocytogenes isolates, 13 (59.10 %), 5 (22.73%), 3 (13.63%) and 1 (4.54%) belonged to serotypes 4b, 1/2a, 1/2b, and 3c, respectively. The genetic determinant tetM was detected in 70% of the tetracycline-resistant isolates. Out of 11 trimethoprim-resistant isolates, 27.27% isolates contained dfrD. Moreover, the ermB gene was found in 83.33% of the erythromycin-resistant isolates. Conclusion: Ampicillin and partly penicillin consider to be suitable antimicrobial agents to treat human listeriosis. Moreover, due to resistance against many antibiotics, it is necessary to continue monitoring and managing antimicrobial resistance.

Download Full-text

Antibiotic resistance profiles in cultivable microbiota isolated from some romanian natural fishery lakes included in Natura 2000 network

BMC Veterinary Research ◽

10.1186/s12917-021-02770-8 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Veronica Lazăr ◽

Irina Gheorghe ◽

Carmen Curutiu ◽

Ioana Savin ◽

Florica Marinescu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Diffusion Method ◽

Beta Lactam ◽

Bacterial Strains ◽

Great Probability ◽

Integrase Gene ◽

Zoonotic Risk ◽

High Level

Abstract Background The present study aims the characterization of antibiotic resistance phenotypes and encoding genes in bacterial strains isolated from some Romanian aquatic fishery lowland salted lakes. Material/Methods This study was conducted on 44 bacterial strains, mainly belonging to species used as microbiological indicators of fecal pollution isolated from four natural fishery lakes. All strains were tested for their antibiotic susceptibility by disk diffusion method. Simplex and multiplex PCR were performed to identify the β-lactams antibiotic resistance genes (blaNMD, blaOXA−48, blaVIM, blaIMP, blaCTX−M, blaTEM), sulfonamides (Sul1, Sul2), tetracyclines (TetA, TetB, TetC, TetD, TetM), aminoglycosides (aac3Ia), vancomycin (VanA, VanB, VanC), macrolides (ermA, ermB, ermC) as well as the plasmid-mediated quinolone resistance (PMQR) markers (QnrA, QnrB, QnrS), and class 1 integrons (Int1, drfA1-aadA1). Results The Enterococcus spp. isolates exhibited phenotypic resistance to vancomycin (35 %) and macrolides (erythromycin) (75 %); from the vancomycin – resistant strains, 5 % harboured VanA (E. faecalis), while the erythromycin resistant isolates were positive for the ermA gene (E. faecalis − 10 %, E. faecium − 5 %). The Gram- negative rods (GNR) exhibited a high level of resistance to β-lactams: cefuroxime (63 %), cefazolin (42 %), ceftriaxone (8 %), ceftazidime and aztreonam (4 % each). The genetic determinants for beta-lactam resistance were represented by blaCTX−M−like (33 %), blaNDM−like and blaIMP−like (8.33 %) genes. The resistance to non-β-lactam antibiotics was ascertained to the following genes: quinolones (QnrS − 4.16 %); sulfonamides (Sul1–75 %, Sul2–4.16 %); aminoglycosides (aac3Ia − 4.16 %); tetracyclines (tetA – 25 %, tetC − 15 %). The integrase gene was found in more than 50 % of the studied strains (58.33 %). Conclusions The cultivable aquatic microbiota from fishery lakes is dominated by enterococci and Enterobacterales strains. The GNR strains exhibited high levels of β-lactam resistance mediated by extended spectrum beta-lactamases and metallo-β-lactamases. The Enterococcus sp. isolates were highly resistant to macrolides and vancomycin. The high level and diversity of resistance markers, correlated with a high frequency of integrons is suggesting that this environment could act as an important reservoir of antibiotic resistance genes with a great probability to be horizontally transmitted to other associated species from the aquatic sediments microbiota, raising the potential zoonotic risk for fish consumers.

Download Full-text

Prevalence of virulence factor, antibiotic resistance, and serotype genes of Pasteurella multocida strains isolated from pigs in Vietnam

Veterinary World ◽

10.14202/vetworld.2020.896-904 ◽

2020 ◽

Vol 13 (5) ◽

pp. 896-904

Author(s):

Hung Vu-Khac ◽

T. T. Hang Trinh ◽

T. T. Giang Nguyen ◽

X. Truong Nguyen ◽

Thi Thinh Nguyen

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Pasteurella Multocida ◽

Iron Acquisition ◽

Antibiotic Resistance Genes ◽

Diffusion Method ◽

Atrophic Rhinitis ◽

Disk Diffusion Method ◽

Antimicrobial Sensitivity

Aim: The study was conducted to determine the prevalence and characterization of the Pasteurella multocida isolates from suspected pigs in Vietnam. Materials and Methods: A total of 83 P. multocida strains were isolated from lung samples and nasal swabs collected from pigs associated with pneumonia, progressive atrophic rhinitis, or reproductive and respiratory symptoms. Isolates were subjected to multiplex polymerase chain reaction (PCR) for capsular typing, detection of virulence-associated genes and antibiotic resistance genes by PCR. The antimicrobial sensitivity profiles of the isolates were tested by disk diffusion method. Results: All the isolates 83/83 (100%) were identified as P. multocida by PCR: serogroup A was obtained from 40/83 (48.19%), serogroup D was detected from 24/83 strains (28.91%), and serogroup B was found in 19/83 (22.35%) isolates. The presence of 14 virulence genes was reported including adhesins group (ptfA – 93.97%, pfhA – 93.97%, and fimA – 90.36%), iron acquisition (exbB – 100%, and exbD – 85.54%), hyaluronidase (pmHAS – 84.33%), and protectins (ompA – 56.62%, ompH 68.67%, and oma87 – 100%). The dermonecrotoxin toxA had low prevalence (19.28%). The antimicrobial susceptibility testing revealed that cephalexin, cefotaxime, ceftriaxone, ofloxacin, pefloxacin, ciprofloxacin, and enrofloxacin were the drugs most likely active against P. multocida while amoxicillin and tetracycline were inactive. The usage of PCR revealed that 63/83 isolates were carrying at least one of the drug resistance genes. Conclusion: Unlike other parts of the word, serotype B was prevalent among Vietnamese porcine P. multocida strains. The high antibiotic resistance detected among these isolates gives us an alert about the current state of imprudent antibiotic usage in controlling the pathogenic bacteria.

Download Full-text

Degradation of antibiotic resistance genes and mobile gene elements in dairy manure anerobic digestion

PLoS ONE ◽

10.1371/journal.pone.0254836 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0254836

Author(s):

Yi Wang ◽

Pramod K. Pandey ◽

Sundaram Kuppu ◽

Richard Pereira ◽

Sharif Aly ◽

...

Keyword(s):

Antibiotic Resistance ◽

Anaerobic Digestion ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Dairy Manure ◽

Health Concern ◽

Animal Origin ◽

Rrna Gene ◽

Transposase Gene ◽

Integrase Gene

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

Download Full-text

Molecular detection of antibiotic resistance genes in multidrug-resistant Staphylococcus aureus isolates from dog dental plaque

BULGARIAN JOURNAL OF VETERINARY MEDICINE ◽

10.15547/bjvm.2099 ◽

2019 ◽

Vol 22 (4) ◽

pp. 419-427

Author(s):

S. Nouri Gharajalar ◽

M. Onsori

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Multiplex Pcr ◽

Resistance Genes ◽

Dental Plaque ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Diffusion Method ◽

Pcr Assay ◽

Multiplex Pcr Assay

Multidrug resistant Staphylococcus aureus strains are a major health care problem both in humans and animals. In this work we described three multiplex PCR assays for detection of clinically relevant antibiotic resistance genes in S. aureus isolated from dog dental plaques. Thirty dental plaque samples were collected; then cultural, biochemical and molecular tests performed for isolation and identification of S. aureus from samples. The antibiotic susceptibility of the isolates were checked by Kirby Bauer disc diffusion method and the prevalence of antibiotic resistance genes determined using multiplex PCR assay. As a result S. aureus was isolated from 18 dog plaque samples. Fifteen of these isolates were resistant to penicillin. The mecA gene was more prevalent than blaZ among penicillin-resistant bacteria. Ten of the isolates were resistant to tetracycline. The percentage of tetM was higher than tetK among them. Also, 10 of the isolates were resistant to cefazolin among them bla TEM detected in higher rate than blaSHV and blaOXA-1. Hence multiplex PCR assay is a suitable method for detection of antibiotic resistance patterns of S. aureus isolates.

Download Full-text

Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes

Scientific Reports ◽

10.1038/s41598-020-72264-4 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Abdelazeem M. Algammal ◽

Mahmoud Mabrok ◽

Elayaraja Sivaramasamy ◽

Fatma M. Youssef ◽

Mona H. Atwa ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Virulence Genes ◽

Antibiotic Resistance Genes ◽

Health Concern ◽

Diffusion Method ◽

Multi Drug Resistance ◽

Fish Farms ◽

M Gene ◽

Total Prevalence

Abstract This study aimed to investigate the prevalence, antibiogram of Pseudomonasaeruginosa (P.aeruginosa), and the distribution of virulence genes (oprL,exoS, phzM, and toxA) and the antibiotic-resistance genes (blaTEM, tetA, and blaCTX-M). A total of 285 fish (165 Oreochromisniloticus and 120 Clariasgariepinus) were collected randomly from private fish farms in Ismailia Governorate, Egypt. The collected specimens were examined bacteriologically. P. aeruginosa was isolated from 90 examined fish (31.57%), and the liver was the most prominent infected organ. The antibiogram of the isolated strains was determined using a disc diffusion method, where the tested strains exhibited multi-drug resistance (MDR) to amoxicillin, cefotaxime, tetracycline, and gentamicin. The PCR results revealed that all the examined strains harbored (oprL and toxA) virulence genes, while only 22.2% were positive for the phzM gene. On the contrary, none of the tested strains were positive for the exoS gene. Concerning the distribution of the antibiotic resistance genes, the examined strains harbored blaTEM, blaCTX-M, and tetA genes with a total prevalence of 83.3%, 77.7%, and 75.6%, respectively. Experimentally infected fish with P.aeruginosa displayed high mortalities in direct proportion to the encoded virulence genes and showed similar signs of septicemia found in the naturally infected one. In conclusion, P.aeruginosa is a major pathogen of O.niloticus and C.gariepinus.oprL and toxA genes are the most predominant virulence genes associated with P.aeruginosa infection. The blaCTX-M,blaTEM, and tetA genes are the main antibiotic-resistance genes that induce resistance patterns to cefotaxime, amoxicillin, and tetracycline, highlighting MDR P.aeruginosa strains of potential public health concern.

Download Full-text

Susceptibility of Meat Starter Cultures to Antimicrobials Used in Food Animals in Canada

Journal of Food Protection ◽

10.4315/0362-028x-73.5.916 ◽

2010 ◽

Vol 73 (5) ◽

pp. 916-922 ◽

Cited By ~ 3

Author(s):

R. P. CORDEIRO ◽

T. DU ◽

M. R. MULVEY ◽

D. O. KRAUSE ◽

R. A. HOLLEY

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antimicrobial Agents ◽

Antibiotic Resistance Genes ◽

Starter Cultures ◽

Phenotypic Resistance ◽

Negative Results ◽

Susceptibility Tests ◽

Growth Promotants ◽

Or Genes

Lactic acid bacteria (LAB) are extensively used in the food industry for fermentation processes. However, it is possible that these bacteria may serve as a reservoir for antibiotic resistance genes that can be transferred to pathogens, giving rise to public health concerns. Animal operations that use antimicrobials as growth promotants have been linked to the origin of resistance due to the selective effect of low levels of antimicrobial used in this management strategy. The objective of this study was to determine the antimicrobial susceptibilities and mechanisms of resistance for 30 isolates of meat starter cultures commonly used in dry sausage fermentations to 20 antimicrobial agents. Susceptibility tests were performed by broth microdilution using Iso-Sensitest broth (90%, vol/vol) and de Man Rogosa Sharpe (MRS) broth (10%, vol/vol). The results showed that all 30 isolates exhibited resistance to at least three antimicrobials regardless of antimicrobial class while 17 or 30% of strains were resistant to antibiotics in three or six different classes, respectively. The incidence of antimicrobial resistance was higher among Pediococcus pentosaceus and lower for Staphylococcus carnosus strains. Genetic determinants for the lincosamide, macrolide, and tetracycline antimicrobials were not found using PCR. Phenotypic resistance in the absence of known resistance genes found here suggests that other mechanisms or genes might have contributed to the negative results. Further studies are needed to explore the genetic mechanisms underlying the prevalence of antibiotic resistance in Pediococcus species.

Download Full-text