Purpose To explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9. Methods MALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL. Results Citrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4′-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3′)-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34\u00B0C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang. Conclusion We found C. freundii carrying blaIMP–4, blaNDM–1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.

Coexistence of blaIMP-4, blaNDM-1 and blaOXA-1 in blaKPC-2-producing Citrobacter freundii of clinical origin in China

Several efforts have been made to estimate the prevalence of different types of antibiotic resistance in different regions of Nigeria. The study investigated the prevalence of Klebsiella pneumoniae encoding genes for CTX-M, TEM, and SHV-1 extended-spectrum beta-lactamases (ESBL) enzymes isolated from urine samples of non-hospitalized patients with suspected UTI in Lagos State, Nigeria. A 4-month retrospective study, spanning from May 2023 to August 2023, was conducted. K. pneumoniae was isolated from urine samples using standard microbiological methods. The modified Kirby-Bauer disc diffusion method was used to test for antibiotic susceptibility, and Clinical and Laboratory Standard Institute (CLSI) guidelines were followed for interpretation. The double-disk synergy method was used to confirm the phenotypic identity of ESBL producers, and the results were interpreted in accordance with CLSI guidelines. The phenotypic method was utilized for ESBL screening, employing the standard disk diffusion method. The CTX-M ESBL gene, TEM gene, and SHV gene were found using multiplex PCR. Results revealed that 41 K. pneumoniae isolates were resistant to third-generation Cephalosporins; the isolates showed resistance to ceftazidime in 100% of cases and ceftriaxone in 80.49% of cases respectively. It was determined that 8 (19.51%) of the isolates produced ESBL. Among Klebsiella isolates, the frequencies of the SHV, TEM, and CTX-M genes were 31 (75.61%), 4 (9.76%), and 10 (24.39%), in that order. Four isolates (9.67%) had the SHV, TEM, and CTX-M genes, while eight (19.51%) of the isolates possessed both the SHV and CTX-M genes. This investigation demonstrates high frequency, wide range of patterns and coexistence of ESBL genes in K. pneumoniae isolates from outpatient UTI patients. Although several phenotypic tests have been used for ESBL detection, testing directly for the presence of ESBL genes is always more accurate. The majority of the isolates of Klebsiella pneumoniae ESBL producers had the SHV gene identified. 

Prevalence of Esbl Genes in Klebsiella pneumoniae from Individuals with Community Acquired Urinary Tract Infections in Lagos State, Nigeria

The current study was conducted on four isolates of Citrobacter freundii identified previously molecularly and registered in the Global GenBank under the names ALGH1(accession No. OQ703592), ALGH2(accession No. OQ703593, ALGH3 (accession No. OQ703594), and ALGH4(accession No. OQ703595), Antibiotics profile analysis was achieved against 12 antibiotics, all isolates showed 100% resistance to (tetracycline, Doxycycline and oxytetracycline), whereas the isolates were sensitive to Amikacin, Trimethoprim, Nalidixic acid Norfloxaine, Levofloxacine and Ciprofloxacin. Curing experiments of these plasmids were achieved by physical( high temperature), chemical (urea) and natural substances by using both onion and garlic powder in three concentration (1%,5%,10%), urea was effective curing plasmids at concentrations of 200 and 400 µg/ml. 45°C successed in curing resistance plasmids. To our knowledge this study is considered the first one which recorded the using of onion and garlic powder in plasmid curing. As a result, the garlic in both the 5% and 10% concentrations was successful in curing the genes of both doxycycline and Tetracycline, whereas the onion failed in all concentrations. For all ALGH1,ALGH2&ALGH4 strains of the Citrobacter , while sensitivity appeared all bacterial isolate spresented against (Oxytetracycline) while the garlic as a curing agent at a concentration of (10%) resulted in the curing of antibiotic resistance genes (Tetracycline, Oxytetracycline, Doxycycline). This study aimed to cure plasmids from Citrobacter strains by using natural substances of onion and garlic at varying concentrations. 

Curing of Citrobacter spp. Strains Antibiotic Resistance Harboring Plasmids Using Novel Physical, Chemical and Natural Substances

The results showed that all the studied bacterial isolates had multiple resistance to the studied antibiotics before treatment with (5-FU). The current study aimed to investigate the curing activity of the anti-cancer drug (5-FU) and its effect on bacterial resistance to antibiotics. the results of the current study showed variation in plasmid content between different isolates (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli). The results showed that all of the studied isolates lost their plasmids completely after treatment with sub-MIC concentration of the drug (5-FU). However, treatment with (5-FU) resulted in the loss of resistance of all isolates to the antibiotics to which they were resistant before treatment, the antibiotics (AM,ATM,FEP) became more affecting after treatment with the drug against s.aureus and (PRL,FEP,MEM,CIP)against p.aruogenosa compared with the rest of the antibiotics studied. It was concluded in the current study that the anti-cancer drug (5-FU) demonstrated a distinct plasmid curing ability and had a strong effect on the removal of bacterial resistance to antibiotics, which opens the way for the use of this drug in combination with antibiotics as therapeutic combination against the multidrug resistant pathogenic bacteria which may require conducting additional experiments to investigate the activity of this therapeutic combination. 

The Curing Activity of 5-Fluorouracil (5-FU) and Its Role in Reducing The Antibiotic Sensitivity in Some Resistance Bacteria

Electrospun nanofibrous membranes loaded with IR780 conjugated MoS2-nanosheet for dual-mode photodynamic/photothermal inactivation of drug-resistant bacteria

Human-pathogenic bacteria resistant to one or multiple antibiotics have dramatically increased worldwide in the past decades. These bacteria possess great danger, have become a global issue, and it is now impossible to avoid developing strategies for the restoration of treatment options against infections caused by them. This research aims at profiling plasmids of multidrug-resistant bacteria from various clinical specimens such as ear exudate, sputum, urethral swab, wound swab, urine from the catheter, urine, nasal swab, high vaginal swab, stool, eye swab, and blood at Chukwuemeka Odumegwu Ojukwu University Teaching Hospital, Awka, Anambra State, Nigeria. Our investigation used the agar diffusion method for susceptibility tests and identification of multidrug-resistant bacteria before plasmid extraction and gel electrophoresis. A plasmid curing test was performed with 10% sodium dodecyl sulphate. Of the 860 bacteria whose resistance profile was determined, only 42 were multidrug-resistant. These bacteria include Pseudomonas aeruginosa 16 (38.10%), Staphylococcus aureus 12 (28.57%), Escherichia coli 9 (21.43%), and Klebsiella pneumoniae 5(11.90%). The molecular weight of their plasmids ranges between 20.884 kbp and 187.50 kbp. As indicated by the plasmid bands, some bacteria had similar molecular weight while others had no plasmid. The bacterial pattern of the postcuring sensitivity test showed that the bacteria with plasmid bands were cured as they became susceptible to the drugs they were previously resistant to, while the bacteria without plasmid bands remained resistant to the antibiotics. This implies that the latter’s multidrug resistance is nonplasmid mediated. Our analyses highlight the relationship between plasmids and multidrug resistance as well as the role of plasmids in the transmission of drug resistance across bacteria. Thus, in order to lessen the burden that multidrug-resistant bacteria cause and to improve bacterial infections treatments, there should be continued surveillance and periodic research on antibiotic resistance patterns of bacteria from various clinical settings.

/pdf/multidrug-resistance-tracing-by-plasmid-profile-analysis-and-1487d8qm.pdf

Multidrug Resistance Tracing by Plasmid Profile Analysis and the Curing of Bacteria from Different Clinical Specimens

In the quest for a sustainable nematode control strategy, alkaloids of physic nut (Jatropha curcas) plant parts were evaluated for their nematicidal control on the root-gall nematode disease of Okra and its mucilaginous property and nutrient composition in 2019 and 2020 cropping season. The experiment was a 3×6 factorial experiments arranged in Completely Randomized Design with five replications in the screen house. Alkaloids extracted from the seeds, leaves and roots of physic nut plants were applied separately at 0, 1, 2, 3, 4 and 5 ml / potted okra plant inoculated with 1,200 infective (J2) larvae of M. incognita. Results showed that 5 ml of Root and Seed Alkaloids application significantly (P=0.05) reduced root-gall nematode infection to a gall index of 1 and 1.2 (rarely galled) when compared to the untreated control  (4 -severely galled) plants thereby achieving more than 100 percent increase in mucilaginous property of okra over the control for both years of study. Moisture content, fat, calcium and sodium also increased more than the untreated control at 5 ml of root and seed alkaloids. Phosphorus and nitrogen contents were on the other hand rather enhanced by the infection. Neither Okra nor root-gall nematode infection affected ash, magnesium and potassium contents of the fruits. Nevertheless, bioactive active constituent of Physicnut plant parts such as root and seed alkaloids can thus be developed as eco-friendly non-synthetic nematicide for improved yield.

Use of physicnut alkaloids to control root-gall nematode and to improve mucilaginous property and nutrient composition of okra

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Multidrug Resistance Tracing by Plasmid Profile Analysis and the Curing of Bacteria from Different Clinical Specimens

Use of physicnut alkaloids to control root-gall nematode and to improve mucilaginous property and nutrient composition of okra