Bryophyllum pinnatum and Rauvolfia vomitoria downregulatory impact on efflux pump gene expression in neonatal Streptococcus spp.

Antimicrobial resistance, biofilm formation, and molecular detection of efflux pump and biofilm genes among Klebsiella pneumoniae clinical isolates from Northern Jordan

Efflux pumps play a crucial role in the development of antibiotic resistance. The aim of this study was to investigate the relationship between efflux pump gene expression and resistance gene mutations in Helicobacter pylori. Twenty-six clinical strains with varying resistance characteristics were selected for further experiment. Seven susceptible strains were induced to become resistant, and the expression of efflux pump genes and point mutations were recorded. Four susceptible strains were selected to undergo candidate mutation construction, and changes in efflux pump gene expression were detected. Efflux pump knockout strains were constructed, and their effects on preventing and reversing antibiotic resistance gene mutations were assessed. Results showed that the expression of efflux pump genes hefA and hefD was significantly higher in the multidrug-resistant group compared to other groups. During the process of antibiotic-induced resistance, efflux pump gene expression did not exhibit a steady increase or decrease. Strains with the A2143G or A2142G point mutations in 23S rRNA exhibited lower hefA gene expression. Strains with mutations at 87K/91N, 87N/91 G, 87K/91D, or 87N/91Y in gyrA and the 194insertA mutation in rdxA showed higher hefA gene expression compared to the wild-type strain. During the process of antibiotic-induced resistance, the strain with the knockout of the efflux pump gene hefA developed mutations in the 23S rRNA, gyrA, or rdxA genes later compared to the wild-type strain. Knockout of the efflux pump gene could reverse the phenotypic resistance to clarithromycin or metronidazole in some strains but had no effect on reverse resistance gene mutation. This study suggested that different resistance gene point mutations may have varying effects on efflux pump gene expression. Knockout of the efflux pump gene can delay or prevent antibiotic resistance gene mutations to some extent and can reverse phenotypic resistance to clarithromycin and metronidazole in certain strains.

Recent studies have shown that antimicrobial resistance has become a great threat. This study focuses on and investigates the effect of some natural compounds on the expression of efflux pump genes that play an important role in antimicrobial resistance in Pseudomonas aeruginosa. Among the 93 Pseudomonas aeruginosa that were isolated from different clinical samples, 20% were multidrug-resistant, 32% were extensively drug-resistant, and 2% were pandrug-resistant. Identification of the isolates was done using 16S rRNA gene amplification and sequencing. GC-MS analysis of chamomile oil revealed (is-.beta.-Farnesene) as the major component. Minimum inhibitory concentration values of each of apigenin, chamomile oil, and ceftazidime were evaluated for five of the isolates. Bacterial growth curve analysis revealed a decline in bacterial growth after treatment with sub-MIC levels of chamomile oil and apigenin. The gene expression of each mexA, mexB, mexR, and oprM efflux pump gene in the bacterial isolates using quantitative PCR was evaluated in untreated and treated isolates with a sub-MIC value of apigenin and chamomile oil. Chamomile oil significantly reduced the expression of all four genes. On the other hand, apigenin downregulated most genes but upregulated mexA in two isolates and mexB in one isolate. Meanwhile, Chamomile oil exhibited stronger downregulation of efflux pump genes compared to apigenin, with fold changes ranging from 2.28 to 14.3. Accordingly, these novel findings suggest that chamomile oil and apigenin can have a significant effect on inhibiting efflux pump gene expression, thus combating multidrug-resistant P. aeruginosa, and offering new avenues for developing effective natural therapeutic adjuvants to overcome antibiotic resistance. This is preliminary evidence for the gene regulatory potential of chamomile oil and apigenin.

Modulation of the membrane permeability through a decrease in porin-mediated antibiotic entry and/or an increase in antibiotic efflux is one of the resistance mechanisms to antibiotics evolved by Gram-negative bacteria. To assess whether the outer membrane porin OprD and Resistance-Nodulation-Division (RND) efflux pumps were similarly expressed in 33 ciprofloxacin-resistant clinical strains of Pseudomonas aeruginosa and in 30 non-clinical strains originating from the hospital environment (mainly waterborne Pseudomonas aeruginosa), the expression of oprD, mexB, mexF, and mexY genes was investigated. Overall, the expression of oprD was not detected by RT-qPCR in 14 (22%) strains and underexpressed in 35 (56%) more. No significant difference in oprD expression was detected between clinical and non-clinical strains. As for efflux pumps, 23 (70%) of the clinical strains overexpressed at least one of the tested RND genes. Overexpression of mexB, mexF and mexY was detected in 27, 12, and 45% of the clinical strains, respectively. In the 30 non-clinical strains, no overexpression could be found for mexB, mexF, or mexY. On the contrary, a global underexpression of the tested efflux pump genes was recorded. In both clinical and environmental strains, a positive correlation was found between the expressions of oprD and mexB. Similarly, the expressions of oprD and mexF were positively correlated. This result contrasts with the inverse correlation between both MexAB-OprM/MexEF-OprN and OprD previously described in carbapenem-resistant P. aeruginosa strains. The only positive correlation between phenotypic ciprofloxacin minimum inhibitory concentrations (MICs) and the expression of efflux pump gene was witnessed with mexY (analysis on pooled results for clinical and environmental strains). However, in clinical strains, no statistically significant link could be found between the degree of reduction in ciprofloxacin MICs witnessed with Phenylalanine-Arginine β-naphthylamide (PAβN) and the expression of any of the 3 RND genes tested.

1The Review on AMR was an expert panel commissioned by the UK Government in 2014 tasked with analysing the economic and social impacts of AMR and proposing solutions to these. 2NICE is a UK non-departmental public body that sponsored by but separate from the Department of Health that produces evidence-based guidance for health practitioners. 3NHS England is a non-departmental public body sponsored by the Department of Health that oversees planning and delivery of health services in England. 4Note that circulation of the News of the World ceased in July 2011; the Sun on Sunday was launched by the same newsgroup in 2012, but is unavailable on the Nexis database.

Inability of a reserpine-based screen to identify strains overexpressing efflux pump genes in clinical isolates of Staphylococcus aureus

Staphylococcus aureus, a common cause of bovine mastitis, relies on several virulence factors, with biofilm formation being a key contributor to its pathogenicity. The present study investigated the occurrence of S. aureus as etiological agents in bovine mastitis with a focus on the existence of various virulence factors and antibiotic resistance status. Among 120 milk samples collected from West Bengal and Uttar Pradesh, 36 (30%) S. aureus strains were confirmed by conventional methods and PCR. Phenotypic analysis revealed hemolysin (55.55%) and coagulase production (36.11%), while molecular analysis revealed the presence of leukotoxin (luksF, 19.44%), hemolysin (hlb, 58.33%), coagulase (coa, 63.88%), and toxic shock syndrome toxin (tsst-1, 30.55%) genes. Biofilm production ability was detected in 97.22% (crystal violet assay) and 86.11% (Congo red agar assay) strains. Biofilm-associated genes, namely, icaA (80.55%, 29/36), icaB (75%, 27/36), icaC (69.44%, 25/36), icaD (86.11%, 31/36), and MSCRAMMs genes, namely, clfA (58.33%, 21/36), clfB (75%, 27/36), fnbA (75%, 27/36), fnbB (55.55%, 20/36), bap (38.88%, 14/36), bbp (83.33%, 30/36), ebps (69.44%, 25/36), eno (66.66%, 24/36), fib (41.66%, 15/36), and cna (8.33%, 3/36), were also detected. Antimicrobial resistance was observed in 88.88% isolates, with 72.22% exhibiting multidrug resistance (MDR). Among the isolates, 83.33% were methicillin-resistant S. aureus (MRSA), and mecA, femA, and femB genes were present either singly or in combination in 76.66% of the isolates. Efflux pump protein genes, namely, norA, norB, norC, mdeA, mepA, and sepA, were detected either singly or in combination in S. aureus isolates. 61.53% of MDR-MRSA isolates harbored all six efflux pump genes. According to this study, S. aureus of mastitis origin harbors various virulence, antibiotic resistance, biofilm-forming, and efflux pump genes. Bovine mastitis-derived MDR S. aureus isolates can pose a significant public health risk and need urgent attention to formulate strategies for their control and preventing transfer to the human food chain.

BackgroundThe rising antimicrobial resistance (AMR) of Neisseria gonorrhoeae is a major global health concern that limits treatment options and complicates disease management. Efflux pump systems and resistance genes are key to bacteria’s ability to evade antibiotics. This study examined the genetic and phenotypic resistance landscape using a large dataset of whole-genome sequences to identify key resistance mechanisms, assess efflux pump gene prevalence, and analyze regional variations in Minimum Inhibitory Concentration (MIC) values to inform treatment strategies and public health interventions.MethodsA total of 38,585 whole-genome sequences of N. gonorrhoeae were analyzed to identify AMR determinants. This study focused on the presence and distribution of efflux pump genes (mtrC, farB, norM, and mtrA) and specific resistance genes, including tet(C) (tetracycline resistance) and aph(3’)-Ia (aminoglycoside resistance). The MIC values were assessed for multiple antibiotics to evaluate resistance trends and regional variations, including penicillin, spectinomycin, zoliflodacin, gentamicin, and fluoroquinolones.ResultsThis analysis revealed widespread resistance to multiple antibiotics. Efflux pump genes (mtrC, farB, norM, and mtrA) were found in nearly all isolates, highlighting their essential roles in resistance and adaptation. The presence of tet(C) and aph (3’)-Ia varied across different Gene Presence Patterns, suggesting that regional or therapeutic factors may influence tetracycline and aminoglycoside resistance. High MIC values for penicillin were observed, likely because of blaTEM, a beta-lactamase gene responsible for beta-lactam resistance. Resistance to spectinomycin is also widespread, raising concerns about the diminishing efficacy of this antibiotic. In contrast, zoliflodacin, gentamicin, and fluoroquinolones exhibited relatively low MIC values, indicating their sustained effectiveness against N. gonorrhoeae.DiscussionEfflux pump systems are key to N. gonorrhoeae resistance and adaptability. Regional MIC variations indicate that local antibiotic use shapes resistance patterns. The high resistance to penicillin and spectinomycin highlights the need for alternative treatments, whereas zoliflodacin and fluoroquinolones remain effective but require monitoring. This study emphasizes global AMR surveillance, novel therapies, and targeted antimicrobial stewardship to address multidrug-resistant infections.

The stringent response regulators, (p)ppGpp and DksA, modulate various genes involved in physiological processes, virulence, and antimicrobial resistance in pathogenic bacteria. This study investigated the role of DksA in the antimicrobial susceptibility of Acinetobacter baumannii. The ∆dksA mutant (KM0248D) of A. baumannii ATCC 17978 and its complemented strain (KM0248C) were used, in addition to the ∆dksA mutant strain (NY0298D) of clinical 1656-2 strain. The microdilution assay was used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents. Quantitative real-time PCR was performed to analyze the expression of genes associated with efflux pumps. The KM0248D strain exhibited an increase of MICs to quinolones and tetracyclines, whereas KM0248D and NY0298D strains exhibited a decrease of MICs to aminoglycosides. The expression of genes associated with efflux pumps, including adeB, adeI/J, abeM, and/or tetA, was upregulated in both ∆dksA mutant strains. The deletion of dksA altered bacterial morphology in the clinical 1656-2 strain. In conclusion, DksA modulates the antimicrobial susceptibility of A. baumannii. The ∆dksA mutant strains of A. baumannii upregulate efflux pump gene expression, whereas (p)ppGpp-deficient mutants downregulate efflux pump gene expression. (p)ppGpp and DksA conduct opposite roles in the antimicrobial susceptibility of A. baumannii via efflux pump gene regulation.

Neonatal sepsis is still a problem experienced by many newborns. Sepsis in newborns is an invasive bloodstream infection characterized by bacteria found in body fluids such as blood, bone marrow, or urine. The aim of this study was to analyze the relationship between infant weight and neonatal jaundice after neonatal sepsis in newborns. This type of research was a retrospective analytic observational approach The population was all newborns who were treated in the perinatology room at Tarakan City General Hospital. The newborn had been medically diagnosed with neonatal sepsis with neonatal jaundice. The sampling technique used is total sampling. The results showed that the incidence of neonatal jaundice after neonatal sepsis with low birth weight (79.7%) was greater than the normal (60.2%). The analysis obtained a p-value 0.012 so, which means that the p-value <0.05 with a prevalence ratio of 6.357. It means that babies with low birth weight are a risk factor for neonatal jaundice after neonatal sepsis at Tarakan City General Hospital in 2020-2021. The conclusion of this study was that there was a relationship between infant weight and neonatal jaundice after neonatal sepsis in infants. Newborns and babies with low birth weight have six times greater chance of experiencing neonatal jaundice after neonatal sepsis compared to the normal. Keywords: Birth Weight, Neonatal Jaundice, Neonatal Sepsis ABSTRAK Sepsis Neonatal Masih Menjadi Permasalahan Yang Banyak Dialami bayi baru lahir. Infeksi aliran darah invasif yang dikenal sebagai sepsis pada neonatus ditandai dengan adanya bakteri dalam cairan tubuh termasuk urin, cairan sumsum tulang, atau darah. Tujuan penelitian ini untuk menganalisa hubungan berat badan bayi terhadap kejadian ikterus neonatorum setelah terjadinya sepsis neonatorum pada bayi baru lahir. Jenis penelitian ini adalah pendekatan observasional analitik retrospektif. Populasi adalah semua bayi baru lahir yang dirawat di ruangan perinatologi di Rumah Sakit Umum Kota Tarakan dan telah di diagnosis medis sepsis neonatorum dengan kejadian ikterus neonatorum pada bayi baru lahir. Tehnik pengambilan sampel yang digunakan adalah total sampling. Temuan penelitian menunjukkan bahwa kejadian penyakit kuning neonatal setelah sepsis dengan berat badan lahir rendah (79,7%) lebih tinggi dibandingkan kejadian setelah sepsis dengan berat lahir normal (60,2%). Berdasarkan analisis, rasio prevalensi sebesar 6,357 dan p-value <0,05 dengan p-value 0,012. Oleh karena itu, bayi baru lahir dengan berat badan lahir rendah memiliki peluang lebih besar untuk terkena penyakit kuning neonatal setelah adanya kasus sepsis neonatal di RSU Kota Tarakan pada tahun 2020–2021. Kejadian ikterus neonatorum setelah terjadinya sepsis neonatorum pada bayi baru lahir dan bayi dengan berat badan lahir rendah berpeluang mengalami ikterus neonatorum setelah terjadinya sepsis neonatorum 6 kali lebih besar dibandingkan dengan berat badan lahir normal. Kata Kunci: Berat Badan Lahir, Ikterus Neonatorum, Sepsis Neonatorum

The adeABC efflux pump has a crucial role in the resistance of Acinetobacter baumannii strains to antimicrobial agents; it is encoded by adeABC, adeR, adeS genes. We evaluated antibiotic resistance, efflux pump genes, clonal relationships, and analyzed a probable correlation that can exist between antibiotic resistance and the aforementioned genes. We conducted this study on 27 food-originated and 50 human clinical Acinetobacter spp. in Southern Türkiye. MALDI-TOF system and disc diffusion/agar dilution (colistin) methods were used for the identification and antibiotic susceptibility. The efflux pump genes and genetic relatedness of the two groups were investigated by (PCR) and (PFGE) methods. Foodborne A. dijkshoorniae strain was multidrug- resistant (MDR), and none of them resistant to colistin. Most of the clinical isolates (92%) were Extensive-Drug Resistant (XDR); highest resistant to ceftazidime, piperacillin-tazobactam, and imipenem (47, 94%), and were lowest to colistin (7, 14%), respectively. adeABC, and adeR, adeS genes were (23, 85.2%), (9, 33.3%), (27, 100%) and (10, 37.3%), (18, 66.7%) in foodborne strains respectively. These rates were (43, 86%), (48, 96%), (50, 100%), and (34, 68%), (48, 96.7%) in clinical strains respectively. A positive correlation existed between adeA gene positivity and piperacillintazobactam, ceftazidime, gentamycin, imipenem (P=0.048), amikacin (P=0.007) and trimethoprimsulfamethoxazole (P=0.029) resistance in clinical strains. A positive correlation of trimethoprimsulfamethoxazole resistance and adeS gene positivity was seen in foodborne strains (P=0.018). Multiple-efflux pump genes rise in parallel to multidrug-resistance in clinical isolates, while susceptible to diverse antibiotics; food may be a potential provenance for the dissemination of adeABC, adeR and adeS genes.

Infection is a leading cause of neonatal morbidity and mortality worldwide. Premature neonates are particularly susceptible to infection because of physiologic immaturity, comorbidity, and extraneous medical interventions. Additionally premature infants are at higher risk of progression to sepsis or severe sepsis, adverse outcomes, and antimicrobial toxicity. Currently initial diagnosis is based upon clinical suspicion accompanied by nonspecific clinical signs and is confirmed upon positive microbiologic culture results several days after institution of empiric therapy. There exists a significant need for rapid, objective, in vitro tests for diagnosis of infection in neonates who are experiencing clinical instability. We used immunoassays multiplexed on microarrays to identify differentially expressed serum proteins in clinically infected and non-infected neonates. Immunoassay arrays were effective for measurement of more than 100 cytokines in small volumes of serum available from neonates. Our analyses revealed significant alterations in levels of eight serum proteins in infected neonates that are associated with inflammation, coagulation, and fibrinolysis. Specifically P- and E-selectins, interleukin 2 soluble receptor alpha, interleukin 18, neutrophil elastase, urokinase plasminogen activator and its cognate receptor, and C-reactive protein were observed at statistically significant increased levels. Multivariate classifiers based on combinations of serum analytes exhibited better diagnostic specificity and sensitivity than single analytes. Multiplexed immunoassays of serum cytokines may have clinical utility as an adjunct for rapid diagnosis of infection and differentiation of etiologic agent in neonates with clinical decompensation.

To the Editor: We report the first isolation of a multiple antimicrobial drug–resistant strain of Shiga toxin–producing Escherichia coli (STEC) O118:H16 from cattle in Latin America. The strain was isolated during a study of fecal STEC in 205 healthy and 139 diarrheic cattle on 12 beef farms in the state of Sao Paulo, Brazil, in February 2000; one case of STEC was found in a 1-month-old calf with diarrhea. This bovine STEC O118:H16 strain showed resistance to eight antimicrobial substances; the following resistance (R)-genes were detected: ampicillin (blaTEM1-like), kanamycin and neomycin (aphA1), streptomycin (strA/B), sulphametoxazol (sul2), tetracyclin (tet[A]), trimethoprim (no dfrA1, A5, A7, A12, A14, or A17), and trimethoprim/sulphamethoxazol. The STEC O118:H16 strain from Brazil was found to be similar for virulence genes (Shiga toxin 1 [stx1], intimin beta 1 [eae β1], and EHEC-hemolysin [E-hlyA]) and for antimicrobial drug resistance to STEC O118:H16 strains, which were isolated in different countries of Europe (1). Beginning in 1986, STEC O118:H16 was identified as an emerging pathogen for calves and humans in Belgium and Germany (2–4). Cattle and human STEC O118:H16 isolates were similar in virulence attributes and antimicrobial drug resistance and belonged to a distinct genetic clone (1). Transmission of these pathogens from cattle to humans on farms was observed (5). Beginning in 1996, STEC O118:H16 has become important as an emerging pathogen in humans and has been associated with bloody diarrhea and hemolytic uremic syndrome (2). Analysis of the antimicrobial resistance profiles showed that >96% of the European STEC O118:H16 strains showed resistance to one or more antimicrobial drugs in contrast to the 10% to 15% drug-resistant strains that were detected among STEC belonging to other serotypes (1,6,7). STEC O118:H16 showing multiresistance in up to eight different antimicrobial drugs predominated among younger isolates, indicating that drug resistance genes have accumulated over time in STEC O118:H16 strains. The frequency of antimicrobial drug resistance in STEC and Stx-negative E. coli in humans and animals was compared in a study by Schroeder et al. (8). Among human clinical E. coli isolates, antimicrobial resistance was less frequently observed in STEC than in Stx-negative strains, whereas in cattle, antibiotic-resistant strains were found at similar frequencies in both groups of E. coli. The relatively higher frequency of antimicrobial-resistant STEC in cattle was explained by the use of antimicrobial drugs in cattle production, whereas human infections with STEC are generally not treated with antibiotics (8). Cattle could thus be an important source of new emerging antibiotic-resistant STEC strains such as O118:H16. The genetic basis of antimicrobial resistance in STEC O118:H16 is broad, including R-plasmids, integrons, transposons, and chromosomally inherited drug-resistance genes. Fluoroquinolone resistance has also been acquired by some STEC O118:H16 strains (1). The heterogenicity of antimicrobial drug–resistance patterns, the increase of multidrug-resistant strains over time of isolation, and the evidence for multiple aquisition and genetic location of R-determinants indicate that strains belonging to the STEC O118:H16 clone have a propensity to acquire and accumulate R-genes. The finding that multidrug-resistant STEC O118:H16 is isolated from cattle in South America indicates the global spread of this new emerging EHEC type.

Background: With each day, the danger of antibiotic-resistant bacterial strains is increasing worldwide due to the development of resistance mechanisms, including efflux pumps, that are used to expel harmful substances (such as antibiotics) outside bacterial cells. Currently, gold nanoparticles (AuNPs) are regarded as viable substitutes for eradicating microorganisms. Aim of Study: This study aims to investigate the effect of green AuNPs on the gene expression of two efflux pump genes, the norA gene for Staphylococcus epidermidis and the acrA gene for Escherichia coli. Materials and Methods: The green AuNPs were synthesized using pomegranate peel extract and characterized by scanning electron microscope (SEM), ultraviolet–visible spectrophotometry, X-ray diffraction, and Fourier transform infrared spectroscopy. The genes responsible for the expression of multi-drug resistance (MDR) efflux pumps, namely, acrA in E. coli and norA in S. epidermidis, have been identified by polymerase chain reaction (PCR), and the expression of the abovementioned efflux pump genes of both bacteria exposed to different concentrations of AuNPs (minimum inhibitory concentration [MIC] and sub-MIC concentrations) was assessed using RT-PCR. Results: All bacterial isolates of E. coli possess the acrA gene, and those of S. epidermidis possess the norA gene. The results demonstrated a significant downregulation in the expression of efflux pump genes acrA and norA when treated with green AuNPs, with significant differences represented by P value (≥0.05) 0.009 for S. epidermidis and 0.011 for E. coli, as compared to the bacteria untreated with green AuNPs in standardizing with housekeeping genes. Conclusions: Green AuNPs have shown the ability to reduce bacterial resistance by destroying or disabling resistance pathways.

Antimicrobial Resistance (AMR), i.e., the evolution of microbes to become resistant to chemicals used to control them, is a global public health concern that can make bacterial diseases untreatable. Inputs including antibiotics, metals, and biocides can create an environment in the agrifood chain that selects for AMR. Consumption of food represents a potential exposure route to AMR microbes and AMR genes (ARGs), which may be present in viable bacteria or on free DNA. Ready-to-eat (RTE) foods are of particular interest because they are eaten without further cooking, so AMR bacteria or ARGs that are present may be consumed intact. They also represent varied production systems (fresh produce, cooked meat, dairy, etc.). An evidence gap exists regarding the diversity and consumption of ARGs in RTE food, which this study begins to address. We sampled 1001 RTE products at retail sale in the UK, in proportion to their consumption by the UK population, using National Diet and Nutrition Survey data. Bacterial DNA content of sample extracts was assessed by 16S metabarcoding, and 256 samples were selected for metagenomic sequencing for identification of ARGs based on consumption and likely bacterial DNA content. A total of 477 unique ARGs were identified in the samples, including ARGs that may be involved in resistance to important antibiotics, such as colistin, fluoroquinolones, and carbapenems, although phenotypic AMR was not measured. Based on the incidence of ARGs in food types, ARGs are estimated to be present in a high proportion of average diets. ARGs were detected on almost all RTE food types tested (48 of 52), and some efflux pump genes are consumed in 97% of UK diets.