Journal of Global Antimicrobial Resistance

BackgroundTo date, there is a spread of resistance of microorganisms to antibiotics. To solve this problem, the search and development of new drugs with antibacterial activity is necessary. Antimicrobial peptides (AMPs) have pronounced antibacterial activity and may be promising candidates for the role of new drugs. Besides, AMPs can be used to overcome conventional antibiotics resistance due to the possible synergistic effect. In this work, the combined effect of some AMPs (human defensins, HNP-1, hBD-1, hBD-3 and cathelicidin, LL-37) with conventional antibiotics (vancomycin and imipenem) against gram-positive (Enterococcus faecalis; Staphylococcus aureus, methicillin-sensitive, MSSA, and methicillin-resistant, MRSA) and gram-negative (Escherichia coli; Klebsiella pneumoniae; Pseudomonas aeruginosa) bacterial strains was investigated. MethodsBacterial strains were isolated from hospitalized patients of the intensive care unit. Commercially available AMPs (HNP-1, hBD-1, hBD-3, LL-37 by Cloud-Clone Corp., USA) and antibiotics, vancomycin (Sandoz, Slovenia) and imipenem (Merck Sharp and Dohme, USA) were used. Antibiotic resistance phenotypes of isolated bacterial strains were carried out using the disk diffusion method. The standard checkerboard assays were used to study minimum inhibitory concentrations (MIC) of antimicrobials. The combined microbicidal effect of two substances (AMP+conventional antibiotic) was assessed by the fractional inhibitory concentration index (FICI). If FICI [&le;] 0,5, then it was considered that two substances showed synergism of action; if 0.5 < FICI < 4 - no interaction; if FICI > 4 - antagonism. ResultsAll studied AMPs had antibacterial activity against the studied strains. hBD-3 showed the lowest MICs compared to other AMPs. MIC of hBD-3 against S. aureus (MSSA and MRSA), E. coli, K. pneumoniae was the same - 0.5 mg/L, and against P. aeruginosa it was 2 mg/L. The combinations HNP-1+vancomycin (against E. faecalis) and hBD-3+imipenem (against E. coli, K. pneumoniae, P. aeruginosa) according to FICI values have shown the synergistic effect. The results of this study can be used to develop novel antibiotics based on AMPs. Also, in some cases, AMPs can help to overcome resistance to conventional antibiotics.

Background: Antimicrobial resistance (AMR) is a growing global health concern driven largely by inappropriate antimicrobial use. Antimicrobial stewardship programs (ASPs), guided by the Centers for Disease Control and Prevention (CDC) core elements, are essential for optimizing antimicrobial use. However, adherence to these practices and the barriers faced by healthcare workers remain inadequately explored, particularly in resource-limited settings. Objective To assess adherence to the CDC antimicrobial stewardship checklist and identify barriers affecting stewardship practices among healthcare workers at a tertiary care hospital in Uttarakhand, India. Methods A quantitative cross sectional descriptive study was conducted among 355 healthcare workers, including nursing officers and physicians. Data were collected using a sociodemographic questionnaire, the CDC antimicrobial stewardship checklist, and a self-structured barrier assessment tool (test retest reliability r = 0.78). Descriptive and inferential statistics were applied using SPSS version 23.0, with a significance level set at p < 0.05. Results The overall adherence to the CDC antimicrobial stewardship checklist was 52.3%, indicating moderate compliance. Higher adherence was observed in action-oriented interventions, while lower adherence was noted in domains such as accountability, pharmacy expertise, reporting, and education. Major barriers identified included lack of antimicrobial supply (89.0%), shortage of key personnel (88.5%), delays in laboratory reports (85.1%), lack of training (83.9%), and inadequate administrative support (79.2%). Significant associations were found between perceived barriers and factors such as working area, designation, qualification, and work experience (p < 0.05), whereas age and gender showed no significant association. Conclusion Adherence to antimicrobial stewardship practices was moderate, with notable gaps in organizational and educational components. Multiple systemic, resource-related, and behavioral barriers hinder effective implementation. Targeted interventions focusing on strengthening infrastructure, workforce capacity, training, and administrative support are essential to improve stewardship practices in tertiary care settings. Keywords: Antimicrobial resistance, Antimicrobial stewardship program, Barriers, CDC Checklist

Antibiotic self-medication is a form of irrational drug use that contributes to antimicrobial resistance, which results in increasing health care costs and morbidity and mortality rates in the population. The misuse of antimicrobial agents is highly linked with the growing problem of antimicrobial resistance within the population globally. Unless addressed, antibiotic self-medication will drive the world back to the pre-antibiotic era, with people dying helplessly due to infectious diseases. This study aimed to investigate the prevalence of antibiotic self-medication and its associated factors in the Otuke District, Northern Uganda. A community-based cross-sectional study was conducted in the Otuke Town Council, Otuke district. The data of adults aged 18 years and above were collected using a semi-structured questionnaire, and the data were coded and entered into SPSS software version 26. The data were descriptively analyzed for frequencies and percentages. Bivariant and multivariant analyses were performed to determine associations between the variables. Out of 385 participants, 68% self-medicated with antibiotics in the past 12 months. Freedom from drug use (AOR: 3.071; 95% CI: 1.203, 7.876) and unregulated use of antibiotics (AOR at 95% CI: 8.288 (2.815, 24.397)) were more likely to lead to ASM (p value <0.001). Other significant factors included knowledge of antibiotics, previous symptom experience, previous successful treatment, long waiting hours and poor staff attitudes (p value <0.05). The most common self-medicated antibiotics were amoxicillin, Ampiclox and metronidazole. Antibiotic self-medication in the Otuke district is very high due to the availability of medicines and lack of functional drug use regulatory frameworks. The district and government of Uganda should design and implement measures to mitigate this widespread antimicrobial misuse to prevent the development of antimicrobial resistance.

BackgroundAlthough not fully investigated, studies show that Legionella pneumophila can develop antibiotic resistance. As there is limited data available for Portugal, we determined the antibiotic susceptibility profile of Portuguese L. pneumophila serogroup 1 (LpnSg1) isolates against antibiotics used in the clinical practice in Portugal. MethodsMinimum inhibitory concentrations (MICs) were determined for LpnSg1 clinical (n=100) and related environmental (n=7) isolates, collected between 2006-2022 in the context of the National Legionnaires Disease Surveillance Programme, against azithromycin, clarithromycin, erythromycin, levofloxacin, ciprofloxacin, moxifloxacin, rifampicin, doxycycline, tigecycline, and amoxicillin/clavulanic acid, using 3 different assays. Isolates were also PCR-screened for the presence of the lpeAB gene. ResultsTwelve isolates had azithromycin MICs above the EUCAST tentative highest WT MIC, 9 of which were lpeAB negative; for erythromycin and clarithromycin, all isolates tested within the susceptible range. The number of isolates with MICs above the tentative highest WT MIC for the remaining antibiotics was: ciprofloxacin: 7; levofloxacin: 17; moxifloxacin: 8; rifampicin: 11; doxycycline: 82; tigecycline: 4. EUCAST breakpoints are not available for amoxicillin/clavulanic acid. We estimated the ECOFFs and one isolate had a MIC 8-fold higher than the E-test ECOFF. Additionally, a clinical isolate generated three colonies growing on the E-test inhibition zone that resulted in MICs 4-fold higher than for the parental isolate. ConclusionsWe report, for the first time, elevated MICs against first-line and other antibiotics (including azithromycin, fluoroquinolones and amoxicillin/clavulanic acid commonly used to treat pneumonia patients in Portugal) in Portuguese L. pneumophila strains. Results point towards decreased susceptibility in circulating strains, justifying further investigation.

Burn patients are at high risk for nosocomial infection. Antibiotics are the key drugs for the treatment of infections. Overuse and inappropriate use of antibiotics increase both bacterial resistance and the cost of treatment. The introduction of correct and rational use of antibiotics appears to be impossible without having the knowledge of the current situation of antibiotic consumption. So, the study was conducted to know the current situation of antibiotic utilization pattern in burn patients. MethodsA Retrospective review of medical records was done to analyze the utilization pattern of antibiotics. The data were collected from Kirtipur hospital from June 2018 to May 2019. All the admitted patients irrespective of age, gender who were prescribed antibiotics and presented within three days of burn were included in the study. Patients admitted for less than 24hrs of a time were excluded from the study. ResultsA total of 249 reviewed case records came under inclusion criteria. Among them 51.8% were female and 48.2% were male. Mostly affected age group was 15-29 years (34.5%). Flame burn (51.8%) was the main cause of the burn. The majority had second-degree burn and 36.90% had 0-10% burn. Third-generation cephalosporin, ceftriaxone had the highest DDD/100BD (19.05). The most frequently used antibiotics were ceftriaxone, cefazolin, and piperacillin+tazobactam. DU90% comprises 12 antibiotics out of 30 antibiotics. The average number of antibiotics prescribed was 2.12 with a range of 1 to 7. ConclusionsThis study revealed the trend of antibiotic utilization pattern in burn patients. Third-generation cephalosporin, ceftriaxone was the most prescribed antibiotic. Regular antibiotic consumption using DDD methodology is needed for regular monitoring of antibiotic consumption so that timely intervention can be made and this study can be used as a baseline study.

Programmatic Management of multidrug resistant tuberculosis (MDR TB) services were introduced in the Indian TB control programme in 2007. A pharmacokinetic (PK) study of drugs used to treat MDR TB, namely levofloxacin (LFX), ethionamide (ETH), cycloserine (CS), pyrazinamide (PZA), moxifloxacin (MFX) and isoniazid (INH) was undertaken in adult MDR TB patients treated according to the prevailing guidelines in India. Factors influencing drug PK and end-of-intensive phase (IP) status were also determined. We recruited 350 MDR TB patients receiving anti-TB treatment (ATT) in the Indian Government programme in south India. At steady state, serial blood samples were collected, after supervised drug administration. Status at end of IP was noted from the programme records. Of the 303 patients for whom end-of-IP status was known, 214 were culture negative (responders), while 45 patients were either culture positive or required change of regimen or had died before completion of IP (non-responders). The median Cmax (2.0 vs 2.9g/ml; p = 0.005) and AUC0-12 (12.2 vs 17.0g/ml.h; p = 0.002) of ETH were significantly lower in non-responders than responders at IP. In multivariate logistic regression analysis, after excluding defaulters and adjusting for confounders, AUC0-12 of ETH significantly influenced end-of-IP status (aOR - 1.065; 95% CI: 1.001 - 1.134; p = 0.047). Drug doses used currently in the programme produced optimal drug concentrations in majority of patients. ETH played a major role in the MDR TB combination regimen and was a key determinant of end-of-IP status.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant (CR) K. pneumoniae are on the rise in India. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in the present study.K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were tested for antimicrobial susceptibility as recommended by CLSI 2019 and subjected to 96-well microtitre plate biofilm formation assay. Based on optical density at 570 nm isolates were graded as strong, moderate and weak biofilm formers. Subset of strong biofilm formers were subjected to whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Biofilm formation was compared for an association with the carbapenem susceptibility and with patient outcome. Statistical significance, correlations and graphical representation were performed using SPSS v23.0.Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure. The biofilm forming ability had a significant association with the morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (P&lt;0.05) resulted in fewer ‘average days alive’ for the patient (3.33) in comparison to those negative for biofilms (11.33). Phylogenetic analysis including global isolates revealed the clear association of sequence types with genes for biofilm mechanism and carbapenem resistance. Carbapenemase genes were found specific to each clade. The known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2 and wzc with a lack of mutation for hyper-capsulation might be poor biofilm formers. Interestingly, ST15, ST16, ST307 and ST258 – reported global high-risk clones were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, mrkD) and pili (pilQ, ecpA).This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and antimicrobial resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.Competing Interest StatementThe authors have declared no competing interest.View Full Text

1.Background & AimCo-amoxiclav is a commonly used antibiotic. Although, the dose administered during renal replacement therapy may be subtherapeutic. This study aims to describe the current literature on the pharmacokinetics and pharmacodynamics of co-amoxiclav in patientss undergoing renal replacement therapy. MethodWe carried out a systematic review of the available literature in MEDLINE, Embase, Pubmed, and Google Scholar from inception to Oct 2023. Studies were included if they reported pharmacokinetic data on adults given amoxicillin or clavulanic acid during renal replacement therapy. ResultsSeven studies were identified which were published between 1984 to 2021. Variability was observed in the participant characteristics within the studies, the renal replacement therapy settings, the drug exposure, drug assay methods, and the analysis of the pharmacokinetic parameters. ConclusionFurther pharmacokinetic-pharmacodynamic studies are needed on co-amoxiclav during renal replacement therapy.

AimRecently, the rise in Staphylococcal infection incidence accompanied by a rise of antibiotic-resistant strains is a major threat to public health. In this study, mechanisms leading to the occurrence of high-level multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains after fluoroquinolone (FQ) exposure were investigated. MethodologySerially exposing S. aureus ATCC 29213 to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days was performed to obtain S. aureus -1 strains and culturing for another 10 days without antibiotics to obtain S. aureus-2 strains. The genomic alterations in FQ-exposed strains were reached using whole genome sequencing and target sequencing. The expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance were evaluated using RT-qPCR. ResultsAfter serial FQ exposure, we observed a strong and irreversible increase of MICs to all applied FQs, i.e 32 to 128 times in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 significant mutations including 2 deletions, 1 insertion, and 7 missense mutations that occur in all S. aureus-1 and -2 but not in initial strain. The FQ target, GrlA, was also mutated (R570H) in all S. aureus-1 and -2 which can partly explain the development of FQ resistance over the FQ exposure. Besides, FQ exposure also resulted in overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645. ConclusionThe mutations occurred in the FQ-target sequence were associated with high-level FQ resistance while the activation of efflux pump systems and post-translational proteins played an important role in the emergence of MDR in S. aureus. Author summaryAntimicrobial resistance is a major public health problem worldwide. Multiple studies have been performed to understand how bacteria develops resistance during the antibiotic therapy in vitro and in vivo. Here we revealed how Staphylococcus aureus, a stubborn human pathogen, changed its genome and expression of important genes in responding with sub-MIC exposure to flouroquinolone antibiotics. Mutations were found in the target of flouroquinolones such as GrlA (R570H) and interestingly in some hypothetical regions which may be important for gene expression regulation. We have observed an marked overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645 in all exposed strains. Importantly, the expression change still remained when the bacteria were no longer exposed to the antibiotics. This study is important to understand response of S. aureus to flouroquinolone and how it obtains the resistance phenotype under antibiotic exposure.

Background and objectivesAntimicrobial resistance is a major healthcare burden, aggravated when it extends to multiple drugs. While cross-resistance is well-studied experimentally, it is not the case in clinical settings, and especially not while considering confounding variables. In addition, bacteria from different sample sources may have undergone different evolutionary trajectories, therefore examining cross-resistance across sources is desirable. MethodologyWe employed additive Bayesian network (ABN) modelling to examine antibiotic cross-resistance in five major bacterial species, obtained from different sources (urine, wound, blood, and sputum) in a clinical setting, collected in a large hospital in Israel over a 4-year period. ABN modelling allowed for examination of the relationship between resistance to different drugs while controlling for major confounding variables. ResultsPatterns of cross-resistance differed across sample sources. All identified links between resistance to different antibiotics were positive, and most were present in several culture sources. However, in 15 of 18 instances, the magnitudes of the links were significantly different between sources compared. For example, E coli exhibited adjusted odds ratios of gentamicin-ofloxacin cross-resistance ranging from 3.0 (95%CI [2.3,4.0]) in urine samples to 11.0 (95%CI [5.2,26.1]) in blood samples. Conclusions and implicationsOur results highlight the importance of considering sample sources when assessing likelihood of antibiotic cross-resistance and determining antibiotic treatment regimens and policies. Abstract ImportanceWe examine the patterns of antibiotic resistance of a given bacterial species, obtained from different clinical infection locations, while accounting for potentially relevant clinical variables. We find that such patterns of cross-resistance between pairs of antibiotics vary between culture sources (e.g., urine vs blood samples), indicating different selective pressures. These findings have implications on prescription policies aiming to minimize collateral resistance.

BackgroundA multidrug-resistant clinical A. baumannii isolate with resistance to most antibiotics was isolated from a patient at an intensive care unit. The genetic environment, transcriptome, mobile, and resistome were characterized. MethodThe MicroScan system, disc diffusion, and broth microdilution were used to determine the resistance profile of the isolate. A multiplex PCR assay was also used to screen for carbapenemases and mcr-1 to -5 resistance genes. Efflux-pump inhibitors were used to evaluate efflux activity. The resistome, mobilome, epigenome, and transcriptome were characterized. Results & conclusionThere was phenotypic resistance to 22 of the 25 antibiotics tested, intermediate resistance to levofloxacin and nalidixic acid, and susceptibility to tigecycline, which corresponded to the 27 resistance genes found in the genome, most of which occurred in multiple copies through replicative transposition. A plasmid-borne (pR-B2.MM_C3) mcr-1 and chromosomal blaPER-7, blaOXA-69, blaOXA-23 (three copies), blaADC-25, blaTEM-1B, and blaNDM-1 were found within composite transposons, ISs, and/or class 1 and 2 integrons on genomic islands. Types I and II methylases and restriction endonucleases were in close synteny to these resistance genes within the genomic islands; chromosomal genomic islands aligned with known plasmids. There was a closer evolutionary relationship between the strain and global strains but not local or regional strains; the resistomes also differed. Significantly expressed/repressed genes (6.2%) included resistance genes, hypothetical proteins, mobile elements, methyltransferases, transcription factors, membrane and efflux proteins. The genomic evolution observed in this strain explains its adaptability and pandrug resistance and shows its genomic plasticity on exposure to antibiotics.

Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high validity media and biofilm models that mimic chronic wounds, endotracheal tubes, and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections.

Pseudomonas aeruginosa is an opportunistic pathogen with high clinical relevance in intensive care units (ICU) due to its elevated resistance to various antimicrobials, which lead to high morbidity and mortality in patients in critical situations. In this study, we aimed to detect variants of genes encoding {beta}-lactamases and efflux pumps in P. aeruginosa isolates resistant to {beta}-lactams, fluoroquinolones and aminoglycosides. All genes belonging to the subfamilies were included in this study: blaSHV, blaTEM, blaNDM, blaKPC, blaGES, blaCTX-M. In addition, we investigated the most relevant variants of the blaOXA subfamily and genes belonging to the efflux pumps of the Mex family. We tested 54 isolates of P. aeruginosa with a high prevalence of resistance to the antimicrobials piperacillin/tazobactam, ceftazidime, cefepime, imipenem and meropenem. Resistance genes related to carbapenems and spectrum {beta}-lactamases extended were found, which included blaKPC genes (81.49%), blaCTXM-2 (72.22%) and blaCTXM-1 (66.66%). In relation to the presence of Mex family efflux pumps genes, 100% of positivity were detected. These findings suggest that P. aeruginosa isolates exhibit an arsenal of genes encoding {beta}-lactamases able to induce phenotypic patterns of resistance to several antimicrobials commonly used as first-line treatment. Author SummarySince the introduction of the use of antimicrobials, resistance to antimicrobials has been growing and becoming a global public health problem, as it leads to ineffective treatment and an increased risk of mortality. P. aeruginosa is included in the World Health Organization (WHO) critical list of bacteria that have a higher rate of resistance to antimicrobials, requiring constant epidemiological investigation of the strains, especially in hospital environments, to correctly approach them. In this work, we used a methodology that detects 740 variants of different classes of {beta}-lactamases to evaluate the genotype of the study strains against the phenotype found. We evidenced a high prevalence of strains carrying genes related to carbapenems and extended-spectrum {beta}-lactamases, demonstrating a correlation with the phenotypes. Furthermore, we found a 100% positivity rate among the efflux pumps tested belonging to the MEX family.

ObjectivesAcinetobacter baumannii is a Gram-negative nosocomial pathogen that plays an important role in the context of bacterial multidrug-resistance. Increasing resistance to carbapenems in particular is of high therapeutic relevance, as in this case only a few antibiotics remain for treatment. The mechanisms of carbapenem resistance in A. baumannii are mainly based upon carbapenemases and the mechanisms such as porin loss, efflux pumps and altered PBPs have been poorly studied to date. MethodsThe A. baumannii reference strain ATCC 17978 was artificially mutated by selection pressure with increasing meropenem concentrations until carbapenem resistance was achieved. Growth analyses were carried out with the mutants and MICs for relevant antibiotics were determined. In addition, the mutants were whole genome sequenced, and the sequences were compared with the wild type. As various mutagenesis attempts for targeted construction of these respective mutants were unfortunately not successful, the strain collection of the NRC was screened for isolates that showed carbapenem resistance without a detectable carbapenemase. These isolates were sequenced and analysed for abnormalities in PBPs and porins in comparison to the sequence of the reference strain ATCC 17978 and were compared to other strains that possessed a carbapenemase. ResultsIn three of the resulting ATCC 17978 mutants, a mutation of PBP2 was observed (W366L). Theses mutants were carbapenem resistant and were not affected by avibactam in contrast to the wild type ATCC 17978. Growth experiments indicated a fitness loss compared to the wild type. As W366L was not found in the clinical isolates, we looked for other abnormalities in various genes associated with carbapenem resistance. Mutations were primarily found in the PBPs, with a mutation in PBP3 (A515V) occurring particularly often. ConclusionThe results of this work support the prevailing thesis that PBP mutations in A. baumannii can lead to carbapenem resistance. Since there are hardly any studies on this hypothesis and for the most part only using outdated methods, these results are of particular relevance and further studies on this topic are recommended.

The study investigated the spectrum of antibiotic resistance and the associated genes for aminoglycoside, macrolide and ESBL class of antibiotics using clinical isolates. A total of 430 preserved bacterial strains (Acinetobacter baumannii, n= 20; Pseudomonas aeruginosa, n= 26; Klebsiella pneumoniae, n= 42; E.coli, n= 85; Staphylococcus aureus, n= 84; Salmonella Typhi, n= 82; Enterococcus spp., n= 27; Streptococcus pneumoniae, n= 36 and CNS, n= 28) were examined. The strains were isolated from patients admitted to various tertiary hospitals of Dhaka city between 2015 and 2019 with either acute respiratory infections, wound infections, typhoid fever or diarrhea. The isolates were reconfirmed by appropriate microbiological and biochemical methods. Antimicrobial susceptibility tests were done using Kirby-Bauer disk diffusion approach. PCR amplification using resistance gene-specific primers for aminoglycoside, macrolide and ESBL class of antibiotics was done and the amplified products were confirmed by Sanger sequencing. Of the total isolates, 53% came out as MDR with 96.6% of E. coli and 90% of Staphylococcus aureus. There was a year-wise gradual increase of MDR isolates from 2015-2018 and by 2019 the increase in MDR isolates became almost 2-fold compared to 2015. Among the five ESBL genes investigated, CTXM-1 came out as the most prevalent (63%) followed by NDM-1 (22%) and E. coli isolates were the predominant reservoir of these genes. ErmB (55%) was the most frequently detected macrolide resistance gene, whereas aac(6)-Ib (35.44%) was the most prevalent aminoglycoside resistance gene and these genes were most prevalent in E. coli and P. aeruginosa isolates, respectively. CTXM-1 and ErmB (16.66%) were the most frequent partners of coexistence followed by CTXM-1 and aac(3)-II.

Drug-resistant Mycobacterium tuberculosis is one of the leading causes of global mortality. Mechanisms, such as slow uptake of drugs along with cell wall impermeability and active efflux, are some of the concerning reasons leading to drug resistance. Efflux pumps actively transport a wide variety of drugs and toxins away from the target site, which is considered an emerging cause for the failure of anti-tubercular medications and treatment. In this study, we report that the ability of Rv1250, a probable MFS-type transporter, influences the extrusion of multiple structurally unrelated classes of drugs, enhances the biofilm formation in E. coli and Mycobacterium smegmatis, and facilitates the survival of M. smegmatis cells inside the macrophage during antibiotic stress. Interestingly, in trans, the expression of rv1250 decreased the susceptibility of host cells to several structurally unrelated antibiotics, ranging from fluoroquinolones to aminoglycosides, beta-lactams, and anti-tubercular drugs, thus indicating its involvement in imparting intrinsic drug tolerance. In addition, the increased efflux of EtBr, norfloxacin, and Bocillin FL from host cells expressing rv1250 was revealed by the hosts ability to confer a lower level of antibiotic accumulation. Moreover, the expression of rv1250 resulted in the enhancement of biofilm formation. Overall, we conclude that Rv1250 of Mycobacterium tuberculosis might facilitate the survival of host cells under antimicrobial stress. One sentence summaryRole of Rv1250 as an efflux pump

Klebsiella pneumoniae is one of the leading causes of community and nosocomial infections. Reduced treatment options against extensively drug resistant (XDR) - K. pneumoniae, is a serious concern in hospital settings, and hence, WHO has categorized it as a "critical priority pathogen". Biofilm forming ability is a common virulence mechanism amongst K. pneumoniae that is associated with antibiotic tolerance up to 1000X MIC and hence, are difficult to treat. N-acetyl cysteine (NAC) is an FDA approved mucolytic drug used to treat acetaminophen-associated toxicity and obstructive pulmonary diseases. In this study, we assessed NACs antibacterial and antibiofilm activity against clinical isolates of XDR K. pneumoniae, obtained from Madras Medical Mission Hospital, India. To assess the biofilm eradication ability of NAC, we grew biofilms in 96 well plates and treated the mature biofilms with different concentrations of NAC. We observed that the biofilms of only 3 isolates of XDR K. pneumoniae could be eradicated at a concentration as low as 20mg/ml. Although increasing the concentration of NAC to 80mg/mL could significantly reduce the biofilms of all the isolates up to 4-5 Log, NAC at a concentration of 100 mg/mL successfully eradicated the mature biofilms of all the isolates of XDR K. pneumoniae. This in vitro study demonstrates the potential of NAC as an efficient agent against the biofilms of clinical isolates of XDR-K. pneumoniae and thus, provides a promising alternative to antibiotics.

BackgroundEnterobacter spp. are rod-shaped Gram-negative opportunistic pathogens belonging to Enterobacterales. This study aimed at the molecular and genomic characterization of multi-drug resistant Enterobacter spp. isolates recovered from hospitalized patients in a tertiary care hospital in Lebanon. MaterialsA total of 59 Enterobacter spp. clinical isolates consisting of 41 carbapenem-resistant and 18 susceptible by E-test were included in this study. Genotypic identification through whole-genome sequencing was performed and confirmed in silico. Resistance and plasmid profiles were studied using ResFinder4.0 and Plasmid-Finder2.1. Multi-locus sequence typing (MLST) was used to determine the isolates clonality. ResultsANI identified and confirmed that 47 (80%) isolates were E. hormaechei, 11 (18%) were Klebsiella aerogenes and 1 (2%) was an E. cloacae. Carbapenem-resistance was detected among 41 isolates all showing an MIC90 of [&ge;] 32 {micro}g/ml for ertapenem, imipenem, and meropenem. blaNDM-1 (58.5%), blaACT-16 (54%), and blaOXA-1 (54%) were the most common detected {beta}-lactamases, while blaCTX-M-15 gene (68%) was the main detected extended-spectrum {beta}-lactamase (ESBL) encoding gene. Chromosomal ampC gene, carbapenemase encoding genes, and porin modifications were among the detected carbapenem resistance determinants. The carbapenemase encoding genes were linked to three well-defined plasmid Inc groups, IncFII/IncFIB, IncX3, and IncL. MLST typing revealed the diversity within the studied isolates, with ST114 being the most common amongst the studied E. hormaechei. ConclusionThe spread of carbapenem-resistant isolates in clinical settings in Lebanon is a serious challenge. Screening and continuous monitoring through WGS analysis could effectively limit the dissemination of drug-resistant isolates in hospitalized patients. ImportanceDrug resistance is an increasing global public health threat that involves most disease-causing organisms and antimicrobial drugs. Drug-resistant organisms spread in healthcare settings, and resistance to multiple drugs is common. Our study demonstrated the mechanisms leading to resistance against the last resort antimicrobial agents among members of the Enterobacteriaceae family. The spread of carbapenem-resistant bacteria in clinical settings is a serious challenge. Screening and continuous monitoring could effectively limit the dissemination of drug-resistant isolates in hospitalized patients.

Antimicrobial resistance (AMR) presents a significant health problem globally with the majority of the burden coming from lower-middle-income countries. AMR surveillance under a One Health paradigm is critical for determining the relationships between clinical, animal, and environmental AMR levels. Allowing for a thorough knowledge of the interconnected variables contributing to resistance, which enables the development of effective solutions. This systematic review was conducted to determine the impact of antibiotics on the gene expression of Pseudomonas spp. In the East African Community. A comprehensive literature search was conducted across Web of Science, Scopus, and PubMed databases yielding 284 articles with 11 meeting the inclusion criteria after screening. We included the 11 studies from 5 East African Countries that are part of the East African Community, the results revealed a high prevalence of antimicrobial resistance in Pseudomonas aeruginosa, with resistance rates above 90% for most tested antibiotics, exception of Amikacin, which remained effective due to its limited use. Common resistance genes reported included carbapenem-resistant genes like blaNDM-1 and blaVIM, the most common method used was disc diffusion method at (50%). The review also found high-risk clones, such as ST 244 and ST 357, that were associated with multidrug-resistant strains. Environmental isolates showed lower resistance rates (54%) than clinical pathogens (73%), indicating different selecting pressures. Majority of the studies were conducted in Kenya (30%) and Uganda (30%), indicating differences in research capabilities and healthcare facilities. These findings highlight the critical need for more surveillance, effective antimicrobial stewardship programs, and additional research to prevent antibiotic resistance and guide public health initiatives in the region. KEY FINDINGS OF THE STUDYPseudomonas aeruginosa isolates demonstrated substantial resistance to antibiotics, including cefepime, meropenem, levofloxacin, and ticarcillin-clavulanic acid as reported across various studies conducted in East Africa. Amikacin was reported to be more effective in more than 90% of the studies reported across East Africa as a potential treatment choice for multidrug-resistant Pseudomonas infections in the region. Carbapenem-resistant genes such as blaNDM-1, blaVIM, and blaOXA-48 were found in a large number of clinical and environmental isolates. High-risk clones, such as ST 244 and ST 357 were reported to demonstrate clonal spread of multidrug-resistant Pseudomonas aeruginosa across East African healthcare settings. The disc diffusion method was the most popular antimicrobial susceptibility testing method (50%), owing to its low cost and simplicity. DNA extraction and PCR were used in 30% of the studies whereas more advanced approaches such as whole genome sequencing were less popular due to resource constraints. The majority of studies were undertaken in Kenya (30%) and Uganda (30%), with fewer studies in Tanzania and the Democratic Republic of the Congo (20%), demonstrating regional variations in research capacity and healthcare resources.

Pseudomonas aeruginosa has been considered one of the major nosocomial pathogens associated with elevated morbidity and mortality worldwide. Outbreaks have been associated with few high-risk pandemic P. aeruginosa lineages, presenting a remarkable antimicrobial resistance. However, the biological features involved with the persistence and spread of such lineages among clinical settings remain to be unravel. This study reports the emergence of the ST309 P. aeruginosa lineage in South America/Brazil, more precisely, in the Amazon region. Global genomic analyses were performed with the Brazilian strain (PA834) and more 41 complete and draft ST309 genomes publicly available, giving insights about ST309 epidemiology and its resistome and mobilome. Antimicrobial susceptibility tests revealed that the Brazilian PA834 strain presented the XDR phenotype, which was mainly due to intrinsic resistance mechanisms. Genomic analyses revealed a heterogeneous distribution of acquired antimicrobial resistance genes among ST309 genomes, which included blaVIM-2, blaIMP-15 and qnrVC1, all of them associated with class 1 integrons. The mobilome mining showed the presence of Integrative and Conjugative Elements, transposons and genomic islands harbouring a huge arsenal of hevy metal resistance genes. Moreover, these elements also carried genes involved with virulence and adaptive traits. Therefore, the presence of such genes in ST309 lineage possibly accounted for the global spread and persistence of this emerging clone, and for its establishment as a pandemic lineage of clinical importance.