Flagellin deficiency drives multi-drug resistance in Salmonella through biofilm adaptation and efflux pump activation.

Background: The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Pseudomonas aeruginosa represents a significant challenge to current antibiotic therapies, particularly in immunocompromised patients, leading to severe and potentially fatal infections. Objectives: The present study aims to investigate the association between resistance patterns, the presence of the algD, pelF, and pslD genes, and biofilm formation capabilities among clinical isolates of P. aeruginosa. Methods: A total of 25 clinical strains of P. aeruginosa were isolated from clinical specimens. An antibiotic sensitivity test was conducted to categorize the organisms as resistant (R), MDR, and XDR strains. A biofilm formation assay was performed to evaluate biofilm formation capacity, and the algD, pelF, and pslD genes were detected using a polymerase chain reaction (PCR) technique. Results: All 25 clinical isolates showed the highest resistance against meropenem (MEM) (100%), imipenem (IPM) (96%), and ceftazidime (CAZ) (72%). Antimicrobial resistance (12%) and MDR (12%) strains were more prevalent in the 51 - 60 and 71 - 80 age groups, while the highest MDR (16%) strains were detected in the 81 - 90 age group. In males, R strains (28%) were isolated, and in females, the highest MDR (24%) strains were found, with no significant gender differences. In sepsis diagnosis cases, the highest antimicrobial resistance (16%), MDR (16%), and XDR (8%) strains were isolated. The algD gene was detected in R strains (36%), while pelF (40%) and pslD (40%) were detected in MDR strains. Strong biofilm formation was found in MDR (20%) and XDR (12%) strains, and moderate biofilm formation was observed in R strains (28%). Conclusions: Overall, the study found an association between biofilm formation and MDR/XDR strains. This suggests that the highest biofilm formation and percentage of pelF and pslD gene detection in MDR strains may contribute to the persistence and severity of infections caused by these strains.

Pseudomonas aeruginosa is a significant pathogen identified with healthcare-associated infections. The present study evaluates the role of biofilm and efflux pump activities in influencing high-level resistance in virulent P. aeruginosa strains in clinical infection. Phenotypic resistance in biotyped Pseudomonas aeruginosa (n = 147) from diagnosed disease conditions was classified based on multiple antibiotic resistance (MAR) indices and analysed with logistic regression for risk factors. Efflux pump activity, biofilm formation, and virulence factors were analysed for optimal association in Pseudomonas infection using receiver operation characteristics (ROC). Age-specificity (OR [CI] = 0.986 [0.946–1.027]), gender (OR [CI] = 1.44 [0.211–9.827]) and infection sources (OR [CI] = 0.860 [0.438–1.688]) were risk variables for multidrug resistance (MDR)-P. aeruginosa infection (p < 0.05). Biofilm formers caused 48.2% and 18.5% otorrhea and wound infections (95% CI = 0.820–1.032; p = 0.001) respectively and more than 30% multidrug resistance (MDR) strains demonstrated high-level efflux pump activity (95% CI = 0.762–1.016; p = 0.001), protease (95% CI = 0.112–0.480; p = 0.003), lipase (95% CI = 0.143–0.523; p = 0.001), and hemolysin (95% CI = 1.109–1.780; p = 0.001). Resistance relatedness of more than 80% and 60% to cell wall biosynthesis inhibitors (ceftazidime, ceffproxil, augumentin, ampicillin) and, DNA translational and transcriptional inhibitors (gentamicin, ciprofloxacin, ofloxacin, nitrofurantoin) were observed (p < 0.05). Strong efflux correlation (r = 0.85, p = 0.034) with MDR strains, with high predictive performances in efflux pump activity (ROC-AUC 0.78), biofilm formation (ROC-AUC 0.520), and virulence hierarchical-clustering. Combine activities of the expressed efflux pump and biofilm formation in MDR-P. aeruginosa pose risk to clinical management and infection control.

Nontyphoid Salmonella Infection: Microbiology, Clinical Features, and Antimicrobial Therapy

Background: Appearance of multi-drug resistance (MDR) Acinetobacter baumannii imposes limitation on antibiotic therapy in patients. Detection of MDR A. baumannii can play a crucial role to prevent MDR strains spreading in hospitals. The aim of this study was determination the efflux pumps gene expression in tigecyclin resistance strains in collected isolates from selected training hospitals in Tehran, Iran. Materials and Methods: In this cross sectional study, A. baumannii was collected from July to February 2014. Tigecycline susceptibility testing has been prepared according to CLSI guide- lines after identification. Active efflux pumps have been detected by Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as an efflux pumps inhibitor. Gene expressions of these efflux pumps have been determined by Real- Time PCR. Results: In this study 80 A. baumannii have been confirmed by conventional phenotypic methods. Tigecyclin resistant was confirmed according to antibiotic susceptibility testing results. The results of CCCP indicated that 22.5% of tigecycline resistant A. baumannii could include active efflux pumps. The results of Real- Time PCR indicated that abeM gene expression has been observed in the most of CCCP positive A. baumannii and adeB has been observed in the minimum number of strains. Conclusion According to the results of this study,Efflux pumps can play an important role in appearance of cross resistance and make MDR strains. Thus, the detection of antibiotic resistance related to active efflux pumps may be crucial to find a composition with efflux pump inhibitor effect by clinical usage.

An efflux pump (MexAB-OprM) of Pseudomonas aeruginosa is associated with antibacterial activity of Epigallocatechin-3-gallate (EGCG)

Background emergence of multidrug-resistant (MDR) bacterial strains is a public health concern that threatens global and regional security. Efflux pump-overexpressing MDR strains from clinical isolates are the best subjects for studying the mechanisms of MDR caused by bacterial efflux pumps. A Klebsiella pneumoniae strain overexpressing the OqxB-only efflux pump was screened from a clinical strain library to explore reverse OqxB-mediated bacterial resistance strategies. We identified non-repetitive clinical isolated K. pneumoniae strains using a matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry clinical TOF-II (Clin-TOF-II) and susceptibility test screening against levofloxacin and ciprofloxacin. And the polymorphism analysis was conducted using pulsed-field gel electrophoresis. Efflux pump function of resistant strains is obtained by combined drug sensitivity test of phenylalanine-arginine beta-naphthylamide (PaβN, an efflux pump inhibitor) and detection with ethidium bromide as an indicator. The quantitative reverse transcription PCR was performed to assess whether the oqxB gene was overexpressed in K. pneumoniae isolates. Additional analyses assessed whether the oqxB gene was overexpressed in K. pneumoniae isolates and gene knockout and complementation strains were constructed. The binding mode of PaβN with OqxB was determined using molecular docking modeling. Among the clinical quinolone-resistant K. pneumoniae strains, one mediates resistance almost exclusively through the overexpression of the resistance-nodulation-division efflux pump, OqxB. Crystal structure of OqxB has been reported recently by N. Bharatham, P. Bhowmik, M. Aoki, U. Okada et al. (Nat Commun 12:5400, 2021, https://doi.org/10.1038/s41467-021-25679-0). The discovery of this strain will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and builds on the foundation for addressing the threat posed by quinolone resistance.IMPORTANCEThe emergence of antimicrobial resistance is a growing and significant health concern, particularly in the context of K. pneumoniae infections. The upregulation of efflux pump systems is a key factor that contributes to this resistance. Our results indicated that the K. pneumoniae strain GN 172867 exhibited a higher oqxB gene expression compared to the reference strain ATCC 43816. Deletion of oqxB led a decrease in the minimum inhibitory concentration of levofloxacin. Complementation with oqxB rescued antibiotic resistance in the oqxB mutant strain. We demonstrated that the overexpression of the OqxB efflux pump plays an important role in quinolone resistance. The discovery of strain GN 172867 will contribute to a better understanding of the role of the OqxB transporter in K. pneumoniae and promotes further study of antimicrobial resistance.

Normal healthy people are not susceptible to tuberculosis (TB) but immunocompromised and HIV positive patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and amikacin) for TB patients is 6-9 months for normal patients but if Mycobacterium tuberculosis (Mtb) becomes multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in 2018, there were about half a million new cases among which 78% were multidrug resistant TB. This antibiotic resistance is due in part to its ability to survive in the macrophage in our body by entering a non-replicating persistent state. Mtb also contains efflux pumps that increase antibiotic tolerance by pumping out the drugs. Therefore, if the efflux pump activity can be blocked by using efflux pump inhibitors, then it might increase antibiotic susceptibility of the pathogen. In our study, we used Mycobacterium smegmatis (Msm) as a model organism for Mtb and subjected it to a combination of three stresses (low oxygen, low pH and low nutrients) that mimic the physiological stresses in the human body and report that these conditions produced a non-replicating state in Msm. This is the first report of the use of this combination of stresses to produce a non-replicating state in Msm. Our results show that non-replicating Msm became completely tolerant to isoniazid and displayed increased tolerance to rifampin and clarithromycin by nearly 2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to construct a gene knockout mutant lacking two potential ATP-binding cassette transporters to study their functions as drug exporters. However, we were unable to obtain homologous recombination mutants. Further studies on efflux pump inhibitors could potentially enable greater understanding of antibiotic tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the development of novel therapies that shorten treatment time for tuberculosis.

Multidrug resistant (MDR) strains of Acinetobacter baumannii present a serious clinical challenge. The development of antibiotic resistance in this species is enabled by efflux pumps of the Resistance-Nodulation-Division (RND) superfamily of proteins creating an efficient permeability barrier for antibiotics. At least three RND pumps, AdeABC, AdeIJK, and AdeFGH are encoded in the A. baumannii genome and are reported to contribute to antibiotic resistance in clinical isolates. In this study, we analyzed the contributions of AdeABC and AdeIJK in antibiotic resistance and growth physiology of the two MDR strains, AYE and AB5075. We found that not only the two pumps have nonoverlapping substrate specificities, their inactivation leads to specific nonoverlapping changes in gene expression as determined by RNA sequencing and confirmed by gene knockouts and growth phenotypes. Our results suggest that inactivation of AdeIJK elicits broader changes in the abundances of mRNAs and this response is modified in the absence of AdeB. In contrast, inactivation of AdeB leads to a focused cellular response, which is not sensitive to the activity of AdeIJK. We identified additional efflux pumps and transcriptional regulators that contribute to MDR phenotype of clinical A. baumannii isolates.

Two simple, instrument-free, user-friendly methods that can readily be implemented by a routine microbiology laboratory are described for the detection of multi-drug-resistant (MDR) isolates that overexpress efflux pump (EP) systems responsible for the MDR phenotype. The first method employs the universal EP substrate ethidium bromide (EB) at varying concentrations in agar-containing plates upon which the contents of an overnight culture are swabbed as spokes of a wheel. In this method, named the EB-agar cartwheel method, it is assumed that the smallest concentration of EB that produces fluorescence of the bacterial mass represents the highest concentration of EB that the bacteria can exclude. Consequently, as the efflux system(s) of a given MDR clinical bacterial isolate is overexpressed relative to that of a reference strain, the minimal concentration of EB producing fluorescence is significantly greater. A simple formula is provided which affords the ranking of MDR clinical isolates with respect to the degree of their efflux capacity. The second method, which can be used after the first one, determines whether the MDR phenotype is based upon an overexpressed efflux system. This method employs a 24-well microplate with separate wells containing or lacking an efflux pump inhibitor (EPI) and Kirby-Bauer discs that correspond to the antibiotics to which the MDR strain is resistant. After the wells are inoculated with the MDR clinical isolate, the plate is incubated overnight and each well is evaluated by eye for evidence of growth. Comparison of growth to the relevant control enables the observer to determine the following outcomes: no growth produced by the EPI-antibiotic combination (i.e., reversal of antibiotic resistance); reduced growth produced by the EPI-antibiotic combination; no difference in growth, i.e., EPI does not affect the resistance to the given antibiotic. If the first method showed that there was a significant difference between the minimum concentrations of EB in agar that produced fluorescence for the clinical isolate and its reference strain, then one can conclude that if the EPI had no effect on reducing antibiotic resistance, the differences in the EB concentrations that produced fluorescence are probably due to differences in the permeability of the strain to EB, reflecting a downregulation of porins if the clinical isolate is a Gram-negative bacterium.

Sir, The worldwide dissemination of multidrug-resistant (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. To reduce the intracellular accumulation of antibiotics, a key bacterial response is to stringently control membrane transporters involved in the diffusion of drugs through the envelope into the cell and those involved in the expulsion of antibiotics, the so-called ‘in and out’ flux. In Gram-negative bacteria, the outer membrane is an additional barrier to antibiotic penetration. Numerous antibiotics that are active against Grampositive are much less active against Gram-negative bacteria. An attractive approach to circumvent bacterial resistance is the development of a chemosensitizer agent to promote an increase of the internal antibiotic concentration in resistant strains. This agent will be used in combination with classic antibiotics to bypass the mechanical and enzymatic barriers that reduce the intracellular concentration of the active antibacterial drug. Squalamine exhibits interesting antimicrobial activity, suggesting its potential application to combat resistant pathogens. It modifies membrane integrity by increasing permeability, as demonstrated by ATP release and dye entry in Gram-negative bacteria, and it has a moderate cytotoxicity at doses that kill MDR bacterial pathogens. The aim of this study was to evaluate the effect of subinhibitory concentrations of squalamine on the activity of various antibiotic classes against different resistant bacteria. The chemosensitizer activity of squalamine was studied by determining the MICs of various usual antibiotics in the presence of subinhibitory concentrations of squalamine. These concentrations corresponded to 1/5 and 1/10 of the respective squalamine MICs determined for each bacterial strain. A control was included by using the well-described efflux pump inhibitor phenylalanine arginine b-naphthylamide (PAbN) to evaluate the involvement of efflux. The strains were grown on Mueller–Hinton broth (Becton Dickinson) at 378C. The Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa strains used have been previously described. Results were read after 18 h at 378C and the MIC values are the means of three independent experiments. The results for documented E. coli strains exhibiting various porins and efflux pump profiles indicated that, even at the low subinhibitory concentration tested (MIC/10), squalamine had an effect on the activity of various different antibiotic classes (Table 1). A significant reduction in chloramphenicol, tetracycline and ciprofloxacin MICs was observed for AG100 and AG100Atet in the presence of squalamine. AG100Atet is an acrAB2 strain that overproduces several drug efflux pumps and down-regulates porin expression. An effect was also obtained on cefepime susceptibility, indicating that squalamine may favour the uptake of this b-lactam molecule via the permeabilizing effect previously reported. It is worthwhile mentioning that squalamine also increases the activity of tested antibiotics against susceptible cells, AG100 and AG100A, demonstrating that the effect is not focused on a resistance mechanism but is related to increased drug penetration. The MICs of erythromycin were not significantly modified by the addition of squalamine, indicating the prominent role of efflux mechanisms for this antibiotic group. In the K. pneumoniae KP55 clinical isolate, the MICs determined in the presence of squalamine were significantly decreased, e.g. 16–32-fold for chloramphenicol, ciprofloxacin, tetracycline and cefepime (Table 1). Squalamine was more efficient at increasing tetracycline and cefepime activity against E. aerogenes than PAbN, highlighting the role of uptake in susceptibility to the two antibiotics. The effect of squalamine on strains devoid of porin (KP63 and KP55) indicated an increase in penetration for cefepime and ciprofloxacin, which normally use porin channels to pass the membrane barrier. Regarding P. aeruginosa and E. aerogenes strains, the results similarly suggest that when the mechanical barrier, restricted uptake or efficient efflux pump participates in resistance, squalamine is able to significantly increase bacterial susceptibility. Squalamine has an effect on the susceptible strains (E. coli, E. aerogenes and P. aeruginosa) tested in this study, suggesting that squalamine may chemosensitize the membrane of nonresistant bacteria and promote the use of a decreased amount of antibiotics. We have previously demonstrated that the alteration of lipopolysaccharide involved in polymyxin-resistant clinical isolates only moderately changes squalamine susceptibility. Consequently, the use of squalamine against polymyxin-resistant isolates selected during colistin treatment, a therapy that is now proposed for the MDR phenotype, may be an attractive hypothesis for the development of future drug combinations. In the bacterial resistance context, and because of its action and its relative insensitivity to efflux resistance mechanisms, squalamine may be a fruitful partner for the development of combinations, such as b-lactams/b-lactamase inhibitors, to combat MDR pathogens. This use allows us to complete our new strategies by promoting the penetration (influx) of antibiotics, in parallel with inhibitors of efflux pumps andb-lactamase inhibitors. This aspect is especially important, since some recent molecules having an original antibacterial spectrum exhibit restricted diffusion through the Research letters

Purpose : To detect efflux pump activity (EPA) and screening a suspected efflux pump inhibitor (EPI) [1- (3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could help in reducing multi-drug resistance (MDR). Methods : Eighteen isolates, viz, 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus species from various hospital infections of admitted patients were screened for antibiotics susceptibility to 11 classes of antibiotics including oxacillin and β-lactamase production. Efflux pump activity (EPA) was determined by minimum inhibitory concentration (MIC) technique in the presence and absence of TFMBP, the isolates were also screened for MDR genes. Results: All the isolates were resistant to ampicillin (10 μg) and penicillin (10 μg), but sensitive to bacitracin (10 μg). Majority of the isolates were MDR 12/18 (66.7 %), 10 (55.6 %) were inducible β- lactamase producers and 3 (16.7 %) were intrinsic β-lactamase producers. Seven (38.9 %) were resistant to oxacillin and also produced carbapenemase enzyme. Eight (66.7 %) of the 12 MDR isolates gave evidence of EPA with TFMBP. However, no MDR genes were detected. Conclusion : Staphylococcus and Micrococcus species exhibit EPA in antibiotic resistance while a suitable EPI such as TFMBP when combined with specific antibiotics could help combat this menace. Keywords : [1-(3-(Trifluoromethyl)benzyl]-piperazine, Efflux pump activity, Oxacillin resistant S. aureus , Multidrug resistant, Carbapenemase

Prophage, a temperate phage embedded in a bacterium's genome, affect bacterial fitness in multiple ways, including infectivity, toxin secretion, virulence regulation, surface modification, immune evasion, and microbiome competition. Studies have revealed that prophages significantly impact the distribution and spread of virulence and antimicrobial resistance. Lysogenic conversion by prophages introduces novel accessory functions to bacteria, enhancing bacterial fitness, host adaptation and persistence in different econiches. Prophages can also be triggered by stress conditions, such as exposure to ultraviolet (UV) radiation, antibiotics, or other chemical agents. Antibiotic-mediated prophage induction is known to cause a high frequency of prophage mobilization, implying that certain clinical strains carrying virulent prophages may have unintended consequences from antibiotics. For example, the hlb-converting prophage (also known as ϕSa3int) of Staphylococcus aureus encodes exotoxins and immune modulatory molecules that can inhibit human innate immunity, increasing the bacterium's pathogenicity. This property contributes to chronic infections and inflammation, such as chronic rhinosinusitis (CRS). Moreover, sub-lethal concentrations of fluoroquinolones, trimethoprim, and β-lactams are known to trigger prophage induction in S. aureus, accelerating the dissemination of prophage-encoded virulence factors to avirulent strains. In this study, our first goal was to identify and describe prophages found in S. aureus isolates recovered from CRS patients and to examine their relationship to CRS disease phenotype and severity. We also aimed to determine whether these prophages could produce active reinfecting phage particles under sub-lethal concentrations of commonly used antibiotics and steroids. Furthermore, we explored links between inducible and non-inducible prophage in S. aureus and factors such as biofilm formation, metabolic activity, and CRS disease severity. Finally, we investigated the genomic and phenotypic plasticity of S. aureus and changes in its extracellular proteome following the acquisition of the ϕSa3int prophage, which was one of the most frequently found prophages in CRS with nasal polyp (CRSwNP) patients. To achieve our goals, we first used various computational tools to identify prophage regions in S. aureus genomes (N = 66) primarily isolated from CRS patients' sinonasal cavities. We then detected virulence and antibiotic resistance genes within the prophage regions of these bacteria. To measure the disease severity of CRS patients, we used computed tomography Lund Mackay scores. We determined antibiotic resistance patterns using the broth microdilution method and identified the minimum inhibitory concentration (MIC). Using sub- MIC concentrations of antibiotics and steroids, we induced the prophages and assessed their infectivity, biofilm biomass and metabolic activity in relation to prophage inducibility. Furthermore, we observed the beta-hemolysis activity of the isolates on sheep blood agar to understand its prevalence in human-adapted S. aureus. Moving forward, we then, induced a ϕSa3int prophage from one of the high biofilm-forming isolates (SA333) and transduced it into another Sa3int-prophage-free S. aureus (SA222, relatively low biofilm forming) isolate to obtain a laboratory-generated 'lysogen'. We confirmed the successful integration of ϕSa3int prophage into hlb-gene and stable lysogenic conversion by short- and long-read sequencing. We then compared the growth kinetics, biofilm biomass, and metabolic activity between the parent and the laboratory-generated lysogen by establishing growth curves, crystal violet and resazurin assays. Finally, we identified and quantified exoproteins secreted by parent strains and lysogens using mass spectrophotometry to understand the virulence factors encoded and secreted by ϕSa3int prophage carrying S. aureus. All S. aureus clinical isolates obtained from CRS patients (N = 57) and control patients (N = 9) carried at least one prophage (average = 3.6 prophages/isolate), with prophages contributing up to 7.7% of the bacterial genome. Based on the completeness scores, we found that nearly 85% (56/66) of S. aureus clinical isolates had at least one intact prophage that were likely inducible. Prophages belonging to type 3 integrase (ϕSa3int-type) were the most prevalent (40%), followed by ϕSa2int (14%). The prophages harbored a distinct set of virulence genes: ϕSa3int-group often encoded human immune evasion cluster genes like sak, scn, chp, and sea, while ϕSa2int-group often harbored leukocidins like lukE/D. Intact prophages were more frequently found in S. aureus isolated from CRS with nasal polyp (CRSwNP) patients than in CRS without nasal polyp (CRSsNP) patients (p = 0.0021). Similarly, intact prophages belonging to the ϕSa3int-group were more frequent in CRSwNP than in CRSsNP (p = 0.0008). Spontaneous prophage induction (SPI) was observed in around 26% (17/66) of the S. aureus clinical isolates, while mitomycin C dependent induction was observed in almost 52% (34/66) of the clinical isolates. Most of the S. aureus clinical isolates showing prophage induction harbored at least one intact prophage(s). Exposure of exponentially growing bacteria to sub-inhibitory concentrations of antibiotics enhanced the prophage induction compared to SPI in almost 50% of active lysogens. Among antibiotics tested, ciprofloxacin was the most potent prophage inducer inducing prophages from 51% of the isolates, followed by amoxicillin, doxycycline, mupirocin, clindamycin, and azithromycin, all of which enhanced the release of prophage in &amp;gt; 40% of the isolates. There was no correlation between S. aureus harboring active prophages and inactive prophages with their biofilm biomass and metabolic activity. However, the disease severity score of patients harboring inducible prophage within S. aureus was significantly lower, implying the role of active lysogeny in CRS disease. In addition, beta-hemolysin activity was absent in almost 92% of S. aureus isolated from the sinonasal cavities of chronic rhinosinusitis patients. Integration of a ~43.8 kb ϕSa3int prophage in one of the hemolysin-producing clinical isolates (SA222) down-regulated the beta-hemolysin expression, implying the role of Sa3int-type prophages in the disruption of beta-hemolysin production. There was no change in bacterial growth kinetics, biofilm formation, adhesion to primary human nasal epithelial cells, and the metabolic activity in a biofilm after ϕSa3int prophage integration. However, the acquisition of ϕSa3int prophage significantly altered the expression of various secreted proteins, both bacterial and prophage encoded. Altogether, thirty-eight exoproteins were significantly differentially regulated in the laboratory-generated lysogen, compared to its recipient strain SA222. Among these proteins, there was significant upregulation of 21 exoproteins (55.3%), including staphylokinase (sak), SCIN (scn), and intercellular adhesion protein B (icaB), and downregulation of 17 exoproteins (44.7%), including beta-hemolysin (hlb/sph) and outer membrane porin (phoE). Most of the upregulated proteins are known for human immunomodulation which helps S. aureus escape human innate immunity and cause chronic infection. In summary, our research has expanded the understanding of prophage distribution in S. aureus among patients with chronic rhinosinusitis (CRS) and their potential impact on the development of the disease. We identified diverse types of prophages in S. aureus within a limited geographic area and among a specific population suffering from CRS. This suggests the presence of a range of prophages that contribute to the adaptability and virulence of the bacteria. Our findings also shed light on the prevalence of active lysogeny in clinical S. aureus isolates and the impact of commonly used antibiotics on prophage mobilization. This can affect both virulence and the spread of antimicrobial resistance. Therefore, our research underscores the importance of minimizing the unnecessary use of antibiotics and the potential hazards associated with exposure of bacteria to sub-lethal antibiotics. Such exposure can promote not only antimicrobial resistance but also accelerates the development of virulent strains. Finally, we also caution against poly-lysogeny, which can worsen the pathogenicity of an isolate through an accumulation of auxiliary phage-encoded traits.

Expression of bacterial efflux pump is one of the major causes for developing antibiotic resistance in a variety of pathogens. Efflux pump inhibitors (EPIs) are potential molecules that can antagonize those pumps and help in alleviating the resistance problem. The study was aimed to explore the EPI activity of vitexin 2"-O-xyloside, an apigenin flavone glycoside. A total 15 clinical isolates of Staphylococcus aureus were collected and their antibiotic resistance profiles were detected by Kirby-Bauer disc diffusion assay and MIC values were determined through broth microdilution technique. Prevalence of efflux pump activity were examined through ethidium bromide agar-cartwheel method. Fractional inhibitory assay was carried out in combination with tetracycline and ciprofloxacin against clinical isolates of S. aureus with efflux pump activity. Finally, molecular docking approach was carried out in the active binding sites of NorA efflux pump protein by Autodock Vina. All of the clinical isolates showed resistance to cefixime, ciprofloxacin and tetracycline antibiotics whereas all of them were sensitive to chloramphenicol. Efflux pump was found active among 20% of the clinical isolates. The tested compound showed additive effective (ƩFIC value 0.625 – 0.75) when co-treated with tetracycline in the efflux pump active isolates which was similar to reserpine. Molecular docking studies showed that vitexin 2"-O-xyloside may bind to the different binding sites, opening the door for it to be considered as a potential EPI. Dhaka Univ. J. Pharm. Sci. 20(3): 307-315, 2022 (June) Centennial Special Issue

SummaryDrugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAβN (Phe-Arg β-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.