IN SILICO ANALYSIS ON BINDING ACTION OF TERPENE NATURAL COMPOUNDS FROM APOCYNACEAE FAMILY AGAINST SHV-1 BETA-LACTAMASE FROM KLEBSIELLA PNEUMONIAE

Many infections produced by multidrug-resistant (MDR) Klebsiella pneumoniae are the main cause of death and treatment restrictions worldwide. In K. pneumoniae, the efflux pump system is dangerous in drug resistance. Therefore, this study was designed to investigate the involvement of the AcrA and AcrB efflux pumps in antibiotic resistance in Klebsiella pneumoniae isolated from wound patients. During June 2021-February 2022, 87 clinical isolates of Klebsiella pneumonia bacteria were obtained from wound samples patients consulted to the hospitals in AL-Diwaniyah province, Iraq. The disc diffusion method performed an antibiotic susceptibility test after microbiological/biochemical identification. The polymerase chain reaction (PCR) technique was used to examine efflux genes' prevalence (acrA and acrB). The results showed that resistance to Carbenicillin 72 (82.7%), Erythromycin 66 (75.8%), Rifampin 58 (66.6%), Ceftazidime 52 (59.7%), Cefotaxime 44 (50.5%), Novobiocin 38 (43.6%), Tetracycline 32 (36.7%), Ciprofloxacin 22 (25.2%), Gentamicin 16 (18.3%), Nitrofurantoin 6 (10.3%) in Klebsiella pneumoniae isolates. The PCR procedure revealed that the occurrence of the acrA and acrB genes is 55 (100%) and 55 (100%), respectively. The findings of this investigation show that the AcrA and AcrB efflux pumps play a crucial character in antibiotic resistance in multidrug-resistant Klebsiella pneumoniae bacterial isolates. As a result of the unintentional transmission of antimicrobial resistance genes, precise detection of resistance genes using molecular approaches is required to switch the extent of resistant strains.

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections, classified into carbapenem-sensitive and carbapenem-resistant strains. Understanding the virulence factors and antibiotic resistance of these strains is essential for effective clinical management. This study compared the virulence genes and antibiotic resistance profiles of 50 CSKP and 50 CRKP strains, examining their expression under antibiotic pressure and the mechanisms contributing to their pathogenicity. Virulence genes (rmpA, rmpA2, iucA, iutA, Peg-344, ybts, iroB) were detected in both strains using polymerase chain reaction (PCR). Antibiotic susceptibility testing established minimum inhibitory concentrations (MICs) for key antibiotics. Gene expression analysis was performed with quantitative reverse transcription PCR (qRT-PCR) after 10 days of antibiotic exposure. CSKP strains exhibited significantly higher positivity rates for virulence genes compared to CRKP strains. CRKP strains predominantly expressed resistance genes KPC, SHV, and CTX-M3, whereas no resistance genes were found in CSKP. Antibiotic susceptibility tests showed increased MICs, particularly for ciprofloxacin and imipenem, following antibiotic induction. CSKP demonstrated elevated expression of rmpA and rmpA2, while CRKP showed increased expression of SHV, and KPC after antibiotic exposure. This study highlights the intricate relationship between virulence and resistance in Klebsiella pneumoniae. CSKP strains show strong virulence factor expression, while CRKP strains adapt to antibiotic pressure through altered gene expression patterns. These findings underscore the urgent need for continuous surveillance and innovative therapeutic strategies to combat multidrug-resistant Klebsiella pneumoniae infections.

The study evaluated the evolution of the incidence of infections with Klebsiella in the County Clinical Emergency Hospital of Craiova (SCJUC), Romania. Also, we monitored antibiotic resistance over more than two years and detected changes in resistance to various antimicrobial agents. Our study included 2062 patients (823 women and 1239 men) hospitalised in SCJUC during the period 1st of September 2017 to 30 June 2019. In 458 patients (22.21%) from the 2062 total patients, the collected samples (1116) were positive and from those, we isolated 251 strains of Klebsiella spp. We conducted a longitudinal analysis of the prevalence of Klebsiella spp. over calendar months, which showed a prevalence in surgical wards that ranged between 5.25% and 19.49% in June 2018, while in medical wards the variation was much wider, between 5.15% and 17.36% in April 2018. Klebsiella spp. strains showed significant resistance to Amoxicillin/Clavulanate, Aztreonam and Cephalosporins such as Ceftriaxone, Ceftazidime and Cefepime. We examined the possible link with the consumption of antibiotics in the same month by performing a multiple linear regression analysis. The evolution of antibiotic resistance in Klebsiella was correlated with the variation of resistance in other bacteria, which suggests common resistance mechanisms in the hospital environment. By performing the regression for dependency between antibiotic resistance and antibiotic consumption, we observed some correlations between antibiotic consumption and the development of antibiotic resistance after 1, 2 and even 3 months (e.g., resistance to meropenem was influenced by the consumption in the hospital ward of imipenem 1 month and two months before, but only 1 month before by the consumption of meropenem). The clustering of strains showed filiation between multiresistant Klebsiella spp. strains isolated from specific patients from the ICU. The evolution of prevalence and antibiotic resistance in Klebsiella correlated with the resistance in other bacteria, which suggest common resistance mechanisms in the hospital environment, and also with the consumption of antibiotics.

BackgroundBacteria treated with different classes of antibiotics exhibit changes in susceptibility to successive antibiotic treatments. This study was designed to evaluate the influence of sequential antibiotic treatments on the development of antibiotic resistance in Klebsiella pneumoniae associated with β-lactamase and efflux pump activities.MethodsThe antibiotic susceptibility, β-lactamase activity, and efflux activity were determined in K. pneumoniae grown at 37 °C by adding initial (0 h) and second antibiotics (8 or 12 h). Treatments include control (CON; no first and second antibiotic addition), no initial antibiotic addition followed by 1 MIC ciprofloxacin addition (CON-CIP), no initial antibiotic addition followed by 1 MIC meropenem addition (CON-MER), initial 1/4 MIC ciprofloxacin addition followed by no antibiotic addition (1/4CIP-CON), initial 1/4 MIC ciprofloxacin addition followed by 1 MIC ciprofloxacin addition (1/4CIP-CIP), and initial 1/4 MIC ciprofloxacin addition followed by 1 MIC meropenem addition (1/4CIP-MER).ResultsCompared to the CON, the initial addition of 1/4 MIC ciprofloxacin inhibited the growth of K. pneumoniae throughout the incubation period. The ciprofloxacin treatments (CON-CIP and 1/4CIP-CIP) showed significant reduction in the number of K. pneumoniae cells compared to meropenem (CON-MER and 1/4CIP-MER). The 1/4CIP-CIP achieved a further 1 log reduction of K. pneumoniae, when compared to the 1/4CIP-CON and 1/CIP-MER. The increase in sensitivity of K. pneumoniae to cefotaxime, kanamycin, levofloxacin, nalidixic acid was observed for CON-CIP. Noticeable cross-resistance pattern was observed at the 1/4CIP-CIP, showing the increased resistance of K. pneumoniae to chloramphenicol, ciprofloxacin, kanamycin, levofloxacin, nalidixic acid norfloxacin, sulphamethoxazole/trimethoprim, and tetracycline. The levels of β-lactamase activities were estimated to be 8.4 μmol/min/ml for CON, 7.7 μmol/min/ml for 1/4CIP-CON and as low as 2.9 μmol/min/ml for CON-CIP. Compared to the absence of phenylalanine-arginine-β-naphthylamide (PAβN), the fluorescence intensity of EtBr was increased in K. pneumoniae cells treated at the CON, CON-CIP, and CON-MER in the presence of PAβN. However, the efflux pump activity remained in K. pneumoniae cells treated at the 1/CIP, 1/CIP–CIP, and 1/CIP-MER in the presence of PAβN.ConclusionThe results suggest that the pre-exposed antibiotic history, treatment order, and concentrations influenced the development of multiple antibiotic resistant associated with β-lactamase and efflux pump activities. This study highlights the importance of antibiotic treatment conditions, which would be taken into consideration when new antibiotic strategy is designed to prevent antibiotic resistance.

Antibiotic resistance in Klebsiella pneumoniae (K. pneumoniae) poses a significant public health challenge globally, with Iraq experiencing a notable rise in multidrug-resistant strains. This narrative review evaluates current evidence on antibiotic resistance patterns and genetic mechanisms underlying resistance in K. pneumoniae isolates across Iraq. The review highlights a high prevalence of B-lactam resistance, particularly to penicillins and cephalosporins, largely driven by extended-spectrum B-lactamase (ESBL) genes such as blaSHV, blaTEM, and blaCTX-M. Carbapenem resistance is alarmingly increasing and is mediated primarily by carbapenemase genes, including blaOXA-48, blaNDM, and blaVIM, with the blaKPC gene emerging in select regions. Aminoglycoside resistance remains moderate, with amikacin retaining efficacy, though resistance genes like strA and strB contribute to variability. Quinolone resistance is attributed to chromosomal mutations, efflux pumps, and plasmid-mediated genes such as qnrS and qnrB, with resistance rates varying significantly by region and clinical setting. The dissemination of resistance is further facilitated by efflux pumps, including acrAB, mdtK, and tolC and porin loss (ompK35, ompK36). Despite increased research, gaps remain due to limited sample sizes and regional disparities in individual studies. To combat this escalating threat, the review advocates for a coordinated national surveillance system, stringent antibiotic stewardship, enhanced infection control measures, and wider implementation of molecular diagnostics. Addressing these challenges is crucial to controlling the spread of resistant K. pneumoniae and preserving the efficacy of existing antibiotics in Iraq.

The ATP binding cassette transporter, VmTPT2/VmABCG1, is involved in export of the monoterpenoid indole alkaloid, vincamine in Vinca minor leaves

Integrons are flexible gene-exchanging platforms that contain multiple cassettes encoding accessory genes whose order is shuffled by a specific integrase. Integrons embedded within mobile genetic elements often contain multiple antibiotic resistance genes that they spread among nosocomial pathogens and contribute to the current antibiotic resistance crisis. However, most integrons are presumably sedentary and encode a much broader diversity of functions. IntegronFinder is a widely used software to identify novel integrons in bacterial genomes, but has aged and lacks some useful functionalities to handle very large datasets of draft genomes or metagenomes. Here, we present IntegronFinder version 2. We have updated the code, improved its efficiency and usability, adapted the output to incomplete genome data, and added a few novel functions. We describe these changes and illustrate the relevance of the program by analyzing the distribution of integrons across more than 20,000 fully sequenced genomes. We also take full advantage of its novel capabilities to analyze close to 4000 Klebsiella pneumoniae genomes for the presence of integrons and antibiotic resistance genes within them. Our data show that K. pneumoniae has a large diversity of integrons and the largest mobile integron in our database of plasmids. The pangenome of these integrons contains a total of 165 different gene families with most of the largest families being related with resistance to numerous types of antibiotics. IntegronFinder is a free and open-source software available on multiple public platforms.

Monoterpene indole alkaloids (MIAs) are valuable metabolites produced in numerous medicinal plants from the Apocynaceae family such as Alstonia scholaris, which synthesizes strictamine, a MIA displaying neuropharmacological properties of a potential importance. To get insights into the MIA metabolism in A. scholaris, we studied here both the spatial and transcriptional regulations of MIA genes by performing a robust transcriptomics analysis of the main plant organs, leaf epidermis but also by sequencing RNA from leaves transiently overexpressing the master transcriptional regulator MYC2. These transcriptomic studies notably demonstrated that the first steps of the MIA pathway are successively distributed in the internal phloem associated parenchyma and epidermis, and that MYC2 exert a remarkable transcriptional effect by modulating the expression of around 1000 genes. By combining these distinct datasets, we initiated the search for MIA-related genes encoding CYP71, based on the similarity of expression compared to already known MIA genes. Transient expression of these candidates in Nicotiana benthamiana leaves and yeast notably led to the identification of a related isoform of rhazimal synthase (RHS) capable of converting the MIA precursor geissoschizine into akuammicine, strictamine and 16-epi-pleiocarpamine. Investigating its catalytic mechanism revealed that strictamine results from rhazimal deformylation and that a similar mechanism may also explain 16-epi-pleiocarpamine synthesis. This prompted us to rename these enzymes geissoschizine cyclase due to their capacity of cyclizing geissoschizine into three different MIA scaffolds and to form both C-C and C-N bonds. This identification thus illustrates the potential of integrating spatial and transcriptional regulation analysis for MIA gene identification.

Helicobacter pylori infection, a leading cause of gastric ulcers and gastric cancer, presents a major health challenge, exacerbated by rising antibiotic resistance. This study investigated the antibacterial potential of plant-derived compounds, isolated from different plant species, against H. pylori. Thus, a library of 153 natural compounds and derivatives, including monoterpene indole and bisindole alkaloids, obtained from the African medicinal plant Tabernaemontana elegans was screened in vitro for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori. Active compounds (1–7) were tested for anti-biofilm activity and cytotoxicity on VERO cells to determine their half-maximal cytotoxic concentrations (CC50). Six monoterpene indole alkaloid azine derivatives (1–6) and vobasinyl-iboga type bisindole alkaloid (7) displayed antibacterial activity, with MICs between 10 and 20 µM. Compounds 2, 3, and 6 exhibited bactericidal activity, with MBCs of 20 µM. Notably, compounds 1 to 4 inhibited H. pylori biofilm formation at sub-inhibitory concentrations. Cytotoxicity assays revealed CC50 values above MICs, indicating a favorable safety profile for potential therapeutic use. This study highlights the potential of T. elegans monoterpene indole alkaloids as antibacterial agents and supports further exploration of plant-derived compounds as alternative treatments for H. pylori, offering a promising approach to address antibiotic resistance in gastrointestinal diseases.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

Klebsiella pneumoniae is by far one of the most common Enterobacteriaceae associated with hospital-acquired infections. The dissemination of multi drug resistant Klebsiella pneumoniae is causing difficulty to treat infections worldwide. Of additional concern, multi drug resistant Klebsiella pneumoniae acquires and transfers antibiotic resistance genes among other bacterial isolates. Integrons have the main role in the acquisition as well as dissemination of resistance genes. Accordingly we aimed to investigate the frequency of resistance genes sul1, sul2, tetA, tetB and aac (3) IIa, class one (int1 gene) and class two integrons(int2 gene) in Klebsiella pneumoniae clinical isolates from four major hospitals in Alexandria, Egypt using Polymerase Chain Reaction. In addition we aimed to evaluate the association between multidrug resistance and presence of integrons in hospital-acquired Klebsiella pneumoniae in our hospitals. To the best of our knowledge, there is little information about integrons and acquisition of multiple antibiotic resistance in Klebsiella pneumoniae in hospitals in Alexandria, Egypt. In this study 76 isolates were resistant to sulphamethoxazole /trimethoprim. Of these 38 isolates (50%) harbored both genes sul1 and sul2 genes. 42 isolates out of the 60 (70%) isolates that showed resistance to tetracycline were tetA or tetB positive. The prevalence of int1 gene among all isolates tested was 90%, while only one isolate harbored the int2 gene (1%). Our results were indicative of the high prevalence of multidrug resistant Klebsiella pneumoniae as well as integrons that were found to play an essential role in the dissemination of antibiotic resistance genes in our hospitals.

Madagascar periwinkle (Catharanthus roseus, family Apocynaceae) is a reservoir of more than 130 monoterpene indole alkaloids (MIAs) including the famous anti-neoplastic dimeric MIAs vinblastine and vincristine, and anti-hypertensive monomeric MIAs ajmalicine and serpentine. Understanding the biosynthetic steps and regulatory factors leading to the formation of MIAs is crucial for rational engineering to achieve targeted enhancement of different MIAs. Due to its highly recalcitrant nature, C. roseus is considered genetically non-tractable for transformation at the whole-plant level. Though few reports have demonstrated tissue culture-mediated regeneration and transformation of C. roseus at whole-plant level recently, the efficiency and reproducibility of these protocols have been a major challenge. To overcome this, we have developed a tissue-culture-independent Agrobacterium-mediated in planta transformation method in C. roseus. Using this method, we were able to efficiently generate stable transgenic plants without relying on the cumbersome methods of tissue-culture regeneration and transformation. Moreover, the transformed plants obtained through this in planta method exhibited stability in subsequent generations. Our method is useful not only for the elucidation of biosynthetic and regulatory steps involved in MIA formation through transgenic plant approach but also for metabolic engineering at the whole-plant level in C. roseus.

Due to the broad-spectrum antibiotic usage and empirical treatments, the pathogenic bacterium, Klebsiella pneumoniae, has shown extremely high detection rates at hospitals with an increasing antibiotic resistance. Therefore, rapid detection of the antibiotic resistance is urgently required and essential for effective treatments. In this study, we evaluated the performance of a newly developed method for ultra-rapid detection of antibiotic resistance in 30–60 min in K. pneumoniae by using the SYBR Green I and propidium iodide (PI) staining. A total of 100 clinical isolates were tested for antibiotic resistance using four different antibiotics (ceftriaxone, cefepime, meropenem, and ciprofloxacin). The results showed that the SYBR Green I/PI rapid antibiotic susceptibility test (AST) could reliably detect antibiotic resistance to the four drugs in 60 min, and the results were highly concordant with the conventional AST (i.e., Kirby-Bauer method and broth microdilution method) for detection of ceftriaxone, cefepime, meropenem, and ciprofloxacin resistance with a high accuracy of 99, 96, 96, and 93%, respectively. Therefore, the rapid AST established in our study helps to enable targeted therapy to save lives and reduce the empirical use of antibiotics and ultimately the health and economic burdens of antibiotic resistance.

BackgroundRecent studies have shown promise in predicting clinical antibiotic resistance rates from sewage data. Few have focused on Klebsiella pneumoniae, despite its virulence and importance as carrier of antibiotic resistance. Several media have been suggested for the isolation of K. pneumoniae from complex samples. However, comprehensive evaluations of culture protocols for isolation of K. pneumoniae from sewage are lacking. MethodsHere, influent samples from a major Swedish sewage treatment plant were used to evaluate ten culture conditions in parallel: cultivation on Brilliant green containing Inositol-Nitrate-Deoxycholate agar (BIND), Bruce agar, Klebsiella ChromoSelect Selective agar®, MacConkey-Inositol-Carbenicillin, or Simmons Citrate Agar with Inositol (SCAI) incubated at either 37°C or 42°C for 44 h. The culture conditions were compared based on colony counts of presumed K. pneumoniae and identification precision assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ResultsThe sensitivity was lowest for BIND, whereas it was similar for the other media irrespective of incubation temperature. For four media, a better precision was observed after incubation at 42°C compared to 37°C, to a large extent explained by a lower frequency of captured Klebsiella oxytoca. SCAI incubated at 42°C showed the highest precision (84.4%). By combining this protocol with subsequent antibiotic resistance screening of collected isolates, low resistance rates in sewage K. pneumoniae were revealed, potentially reflecting the local resistance landscape. ConclusionWhen combined with downstream analyses, SCAI incubated at 42°C could be a valuable culture protocol for sewage-based studies on various aspects of K. pneumoniae epidemiology including antibiotic resistance prevalence.

Every year, the number of deaths caused by antibiotic-resistant infections continues to rise. It is predicted that by 2050, antibiotic-resistant bacteria will surpass cancer as the leading cause of death. The aim of this research is to assess the prevalence of antibiotic resistance among Klebsiella pneumoniae strains in hospitalized patients across Iran. To address this issue, we conducted a thorough search of electronic databases, including PubMed, Scopus, and Web of Science, on March 13, 2023. Using the random-effects meta-analysis model and assessing heterogeneity with Cochran's Q test and I2 statistic, we estimated the prevalence of antibiotic resistance in Klebsiella pneumoniae. Our analyzes were done by STATA v 14. 21 articles met the inclusion criteria. In total, 20 studies were included in the final analysis. The number of studies obtained for ceftazidime, cefotaxime, gentamicin, ciprofloxacin, and imipenem were 17, 13, 13, 14, and 14, respectively. The sample sizes for each were 2,200, 1,557, 1,896, 2,026, and 1779. The pooled estimated prevalence of resistance to Ceftazidime, Cefotaxime, Ciprofloxacin, Gentamicin and Imipenem in Klebsiella pneumoniae were 60%, 63%, 46%, 48% and 26%. These findings highlight that Klebsiella pneumoniae strains in Iran have shown high resistance to commonly used antibiotics, particularly third-generation Cephalosporins which can significantly impact the treatment of infectious diseases. Therefore, it is advisable to explore alternative antibiotics for treating infections caused by this microorganism.