Methicillin-resistant Research Articles

Efflux-mediated Multidrug Resistance in Critical Gram-negative Bacteria and Natural Efflux Pump Inhibitors.

Multidrug Resistance mechanisms in microorganisms confer the slackness of the existing drugs, leading to added difficulty in treating infections. As a consequence, efficient novel drugs and innovative therapies to treat MDR infections are necessarily required. One of the primary contributors to the emergence of multidrug resistance in gram-negative bacteria has been identified as the efflux pumps. These transporter efflux pumps reduce the intracellular concentration of antibiotics and aid bacterial survival in suboptimal low antibiotic concentration environments that may cause treatment failure. The reversal of this resistance via inhibition of the efflux mechanism is a promising method for increasing the effectiveness of antibiotics against multidrug-resistant pathogens. Such EPI, in combination with antibiotics, can make it easier to reintroduce traditional antibiotics into clinical practice. This review mostly examines efflux-mediated multidrug resistance in critical gram-negative bacterial pathogens and EPI of plant origin that have been reported over previous decades.

Essential oils cause membrane disruption and autoaggregation of MDR Acinetobacter baumannii cells

Essential oils are promising antimicrobial agents against various bacteria. The aim of the study was to examine anti-Acinetobacter baumannii activity of nine essential oils and to elucidate essential oil mechanisms of action against this. In total, 8 out of 9 examined different essential oils exhibited activity against multidrug-resistant (MDR) A. baumannii isolates. The best anti-A. baumannii activity expressed Thymus serpyllum, Satureja hortensis and Oreganum majorana essential oil (MBC 1.29 to 2.58 µg mL−1), while essential oils of Menta x piperita, Hyssopus officinalis, Foeniculum vulgare and Artemisia dracunculus expressed the same effect in higher concentrations (1.80 to 3.80 µg mL−1). The effect of Juniperus sabina, Juniperus sibirica and S. hortensis essential oils against A. baumannii is for the first time reported here. The mechanisms of Myrtus communis, Eucalyptus camaldulensis, S. hortensis and T. serpyllum essential oils were further examined. The essential oil treatments led to concentration dependent leakage of biomolecules form the cells, causing an increase in the concentrations of lipids, carbohydrates, and proteins in the suspensions. Microstructural observations confirmed the essential oil effect on cell membranes and disruption the membrane integrity, with obvious leakage of intercellular substance, collapsed cells with perforations, debris presence and autoaggregated cells. The subinhibitory concentrations of oils did not obviously changed protein patterns determined by SDS-PAGE. These results indicate that the essential oils, particularly T. serpyllum, S. hortensis, M. communis and E. camaldulensis express its anti-A. baumannii activity via membrane disruption and increased membrane permeability, representing very promising alternative antibacterial agents against MDR wound isolates.

Antibiofilm and antivirulence efficacy of Pleurotus platypus methanolic extract against Staphylococcus aureus through ROS generation and cell membrane disruption

Multi-drug resistance is recognized as a significant worldwide public health concern in the current century. Biofilm formation further exacerbates bacterial resistance to antibacterial medications, host immunological responses, and phagocytosis, resulting in long-lasting chronic illnesses. Investigating natural resources is a very potent approach for developing alternative anti-infective medications to effectively control multi-drug resistant bacterial infections. In this study, a unique mushroom species namely Pleurotus platypus had been discovered from the Terai-Duars region of West Bengal, India. The myco-chemical profiling and preliminary chemical analysis of Pleurotus platypus methanolic extract determined the significant presence of metabolites belonging to several major chemical classes such as flavonoid, alkaloid, triterpenoid, polyphenol, benzoic acids, coumarin, flavone etc. Most intriguingly, the extract possessed effective antibacterial, antibiofilm and antivirulence properties against Staphylococcus aureus and Methicillin resistant Staphylococcus aureus, one of the most notable drug-resistant opportunistic and nosocomial pathogens. Mechanistically, the mushroom extract enhanced the production of Reactive Oxygen Species (ROS) inside the targeted bacteria, causing alterations in membrane potential, damage to the cellular membrane and further release of intracellular DNA, destined to cell death. Moreover, the methanolic extract reported the eradication of pre-existing biofilms from the urinary catheter surface, hinting towards its future application in the related field. To summarize, Pleurotus platypus methanolic extract could be an excellent alternative antibacterial and antibiofilm therapeutic candidate for the effective management of Staphylococcus infections with improved outcome.

Cancer Stem Cells and Treatment of Cancer: An Update and Future Perspectives.

Cancer stem cells (CSCs) play an essential role in tumour progression and metastasis. Stem cell ability of self-renewal enables it to persist over time, thereby contributing to cancer relapse or recurrence and also resistance to current therapies. Therefore, targeting CSCs emerged as a promising strategy of cancer treatment. CSCs exhibit differentiation, self-renewal, and plasticity, they contribute to formation of malignant tumours, also favors, metastasis, heterogeneity, multidrug resistance, and radiation resistance. Coventional cancer treatments predominantly target cancer cells that are not CSCs, CSCs frequently survive, eventually leading to relapse. This article focuses on the development of novel therapeutic strategies that combine conventional treatments and CSC inhibitors to eradicate cancer cells and CSCs, for the better and permanent treatment. However, the diversity of CSCs is a significant obstacle in the development of CSC-targeted therapies, necessitating extensive research for a better understanding and exploration of therapeutic approaches. Future development of CSC-targeted therapies will rely heavily on overcoming this obstacle.

Molecular insights into antimicrobial resistant Staphylococcus aureus strains: A potential zoonosis of goat origin

Antimicrobial-resistant (AMR) Staphylococcus aureus (S. aureus) strains have attained global attention due to their life-threatening zoonotic nature. Being a member of ESKAPE group, S. aureus has an ability to escape the biocidal action of antimicrobial drugs. The current study investigated the prevalence and molecular characterization of methicillin-resistant S. aureus (MRSA), β-lactam-resistant S. aureus (BRSA), aminoglycoside-resistant S. aureus (ARSA), tetracycline-resistant S. aureus (TRSA), and fluoroquinolones-resistant S. aureus (FRSA) associated with goat subclinical mastitis (SCM). Furthermore, the antimicrobial resistance and susceptibility profile of various antibiotics and non-antibiotics (NSAIDs, nisin, N-acetylcysteine, vitamin-C) along with their possible role in modulating the antibiotic resistance of MDR isolates was also investigated. A total of 768 goat milk samples were subjected to California mastitis test for SCM followed by bacteriological and molecular characterization of S. aureus. Moreover, in-vitro susceptibility of resistant antibiotics, non-antibiotics, and their combination against MDR S. aureus were conducted through well diffusion and broth microdilution assays. The results depicted that 55.47% and 26.82% of milk samples were positive for SCM and S. aureus, respectively. The molecular assay confirmed 35.92% of isolates as MRSA, 45.63% as BRSA, 50.49% as ARSA, and 32.52% but no isolate was confirmed as FRSA on molecular basis. The multidrug resistance was observed in 62.13% and 47.09% isolates, respectively. Molecular characterized MDR S. aureus revealed high homology of study isolates with the isolates of neighboring countries like India, Korea, Iran, and China. Antimicrobial susceptibility trials on well diffusion assay showed higher efficacy of different non-antibiotics with resistant antibiotics as penicillin with ketoprofen and gentamicin with flunixin meglumine while oxytetracycline with N-acetylcystiene. The synergy testing by checkerboard assay revealed synergistic activity of penicillin with ketoprofen, gentamicin with flunixin meglumine, and oxytetracycline with N-acetylcysteine. The current study highlighted the emergence and spread of AMR S. aureus strains from goat SCM and provided insights into possible drug repurposing of various non-antibiotics to modulate the multidrug resistance of S. aureus which will be helpful in devising the therapeutic options and control measures for this pathogen.

The global burden of enteric fever, 2017–2021: a systematic analysis from the global burden of disease study 2021

Enteric fever is a major public health challenge in developing countries. We conducted a systematic analysis from the Global Burden of Diseases 2021 Study to provide updated estimates of enteric fever's burden. We presented estimates for incident cases and deaths, age-standardized incidence and mortality rates, years of life lost (YLLs), and case-fatality rates spanning the study period of 2017-2021, stratified by region, country, socio-demographic index (SDI), and age group. Random-effects Poisson regression for longitudinal data was used to estimate the association between SDI and case-fatality rates, adjusting for antimicrobial resistance patterns. In 2021, there were 9.3 million global cases of enteric fever (95% uncertainty interval: 7.3-11.9) and 107.5 thousand deaths (56.1-180.8). The age-standardized incidence rate decreased from 152/100,000 person-years (118-195) in 2017 to 128/100,000 person-years (100-163) in 2021, and the mortality rate decreased from 1.87/100,000 person-years (0.95-3.18) to 1.50/100,000 person-years (0.78-2.54). There were wide geographical differences, with South Asia contributing the most cases and deaths. Age-standardized incidence exceeded the threshold for "high burden" of enteric fever (100/100,000 person-years) in 23 countries in2021.Children under five accounted for 40% of deaths and 47% of YLLs, with incidence and mortality peaking during the second year. Case-fatality was highest in low SDI countries and showed a global trend toward reduction, except among children aged 1-4 years. After adjusting for the prevalence of multidrug resistance, fluoroquinolone non-susceptibility, and third-generation cephalosporin resistance, a higher SDI was associated with a lower case-fatality rate, with a 1.1% (0.7-1.7) reduction for each percentage point increase in SDI. Despite notable improvements, several countries still showed a high burden of enteric fever, which remains a significant global health concern, especially among children under five. Although enhancing water and sanitation systems is crucial, the most significant reductions in the global disease burden are likely to be achieved through broader vaccine coverage. This includes the use of typhoid conjugate vaccines, which are effective in infants and young children and offer extended protection, along with improved data collection and surveillance to guide vaccine distribution efforts across high-incidence areas. This work was partially supported by "Ricerca Corrente" funding from Italian Ministry of Health to IRCCSHumanitas Research Hospital.

Antibiotic profile classification of Proteus mirabilis using Machine Learning: An Investigation into Multidimensional Radiomics Features

Antimicrobial resistance (AMR) presents a significant threat to global healthcare. Proteus mirabilis causes catheter-associated urinary tract infections (CAUTIs) and exhibits increased antibiotic resistance. Traditional diagnostics still rely on culture-based approaches, which remain time-consuming. Here, we study the use of machine learning (ML) to classify bacterial resistance profiles using straightforward microscopic imaging of P. mirabilis for resistance classification integrated with radiomics feature analysis and ML models.From 150 P. mirabilis strains isolated from catheters of patients diagnosed with a CAUTI, 30 % displayed multidrug resistance using the standardized disk diffusion method, and 60% showed strong biofilm activity in microtiter plate assays. As a more rapid alternative, we introduce wavelet-based and regular microscopy imaging with feature extraction/selection, following image preprocessing steps (image denoising, normalization, and mask creation). These features enable training and testing different ML models with 5-fold cross-validation for P. mirabilis resistance classification. From these models, the Random Forest (RF) algorithm exhibited the highest performance with ACC=0.95, specificity=0.97, sensitivity=0.88, and AUC=0.98 among the other ML algorithms considered in this study for P. mirabilis resistance classification.This successful application of wavelet-based feature Radiomics analysis with RF model represents a crucial step towards a precise, rapid, and cost-effective method to distinguish antibiotic resistant P. mirabilis strains.

Bacterial etiology, antibiotic susceptibility pattern, and treatment modality of surgical site infections among post-caesarean delivery women: A 2019 cross-sectional study conducted in Iringa, Tanzania

Background: Surgical site infection (SSI) is a common complication after cesarean section in Tanzania, with high rates of infection and multidrug-resistant isolates reported. Understanding bacterial etiology and antibiotic susceptibility patterns is crucial for effective treatment and prevention. Therefore, this study aimed to determine the bacterial etiology, antibiotic susceptibility patterns, and treatment modalities of SSI among post-cesarean delivery women in the Iringa region. Methods: A cross-sectional study involving 162 women with SSI was conducted across five hospitals in Iringa. Data on demographics were collected using a structured questionnaire, and wound discharge samples were taken for culture and sensitivity testing. Definitive treatment modalities were identified from patients’ files. Data analysis was performed using SPSS version 20, with chi-square tests used to identify associations, and a p-value < 0.05 considered significant. Results: Among 162 participants, 59.7% had superficial SSIs and 35.2% had deep SSIs. Culture positivity was 96.3%, with 53.2% being gram-positive and 37.2% gram-negative. Staphylococcus aureus was the most commonly isolated pathogen (40.1%), with 64.6% of cases being methicillin-resistant (MRSA). Resistance to vancomycin and chloramphenicol among gram-positive isolates was 3.1% and 13.8%, respectively. Non-surgical management was provided to 58% of the participants, while Staphylococcus aureus infections were significantly associated with surgical intervention (OR = 1.38, p = 0.001). Conclusion: Staphylococcus aureus, particularly MRSA, was the most prevalent pathogen in post-cesarean SSIs. Antibiotic resistance was common, and surgical intervention was significantly associated with Staphylococcus aureus infections.

Antibiotic resistance and the search for resistance and virulence genes in Escherichia coli strains isolated from agropastoral farms (Poultry, Cattle, and Fish) In Southern Benin

Resistance of animal-origin enterobacteria to antibiotics is experiencing a concerning global increase, with an escalating impact of extended-spectrum beta-lactamases (ESBL). Finding solutions to this problem requires reliable and diverse data. It is in this context that the present study was initiated to combat infections caused by Escherichia coli in agropastoral farms (poultry, cattle, and fish) in southern Benin. Samples consisting of cattle dung, poultry droppings, as well as water and sludge from fish farms were aseptically collected for various laboratory analyses. A total of 714 E. coli strains were isolated from 1,376 collected samples, with 434 strains from the Atlantique department and 280 strains from the Zou department. The isolation of strains was carried out according to the NF V08-050: 2009 standard. The identified strains were tested against various antibiotics using the Bauer and Kirby disk diffusion method on agar, as recommended by the WHO. Strains resistant to one or more antibiotics were selected for the search for resistance and virulence genes. Furthermore, multidrug resistance of the strains to antibiotics was also observed, particularly in the Beta-lactam class (100% resistance to AMC) and tetracyclines (90% resistance to tetracyclines). Additionally, genotypic detection revealed a total absence of virulence genes, whereas resistance genes such as beta-lactamase Sulfhydryl Variable (bla-SHV), beta-lactamase Temoniera (bla-TEM), and quinolone resistance gene B (qnrB) were more frequently detected at respectively 58%, 82% and 60%.

Novel plasmid-mediated CMY variant (CMY-192) conferring ceftazidime-avibactam resistance in multidrug-resistant Escherichia coli.

The rapid rise of multidrug resistance (MDR) among Gram-negative bacteria has accelerated the development of novel therapies. Ceftazidime-avibactam (CZA) is a novel β-lactam/β-lactamase inhibitor recently approved for the treatment of limited infectious diseases. Here, we describe a novel CMY variant, CMY-192, that confers high-level resistance to CZA. This gene was detected in a clinical MDR Escherichia coli strain (Ec73552) isolated from an outpatient with a community-acquired urinary tract infection who had not received prior CZA treatment. Ec73552 was typed as O101:H9-ST10, a high-risk clone associated with human and animal diseases. Ec73552 was able to colonize the bladder in a mouse model, suggesting that this strain was uropathogenic. CMY-192 shared the highest amino acid identity (98.95%) with CMY-172 and conferred at least a 32-fold increase in CZA MIC (from ≤0.125/4 to 8/4 mg/L) when cloned into a CZA-susceptible E. coli DH5α strain. Knockout of CMY-192 in Ec73552 resulted in a 256-fold reduction in CZA MIC (from 64/4 to 0.25/4 mg/L). CMY-192 was encoded on an IncB/O/K/Z-type plasmid (pCMY192). Conjugation assays confirmed that pCMY192 was self-transmissible, resulting in a 256-fold increase in the CZA MIC of the recipient. Notably, pCMY192 cured in Ec73552 did not confer a growth advantage, while the conjugant exhibited reduced biomass and growth rate, indicating that fitness costs imposed by pCMY192 may have been compensated in Ec73552. Our findings highlight the importance of continuous monitoring of CZA susceptibility to prevent the spread of resistance in clinical settings.

Injecting drug use is a risk factor for methicillin resistance in patients with Staphylococcus aureus bloodstream infections.

We investigated whether injecting drug use was a risk factor for methicillin resistance among inpatients with Staphylococcus aureus bloodstream infections (SABSIs) at an Australian health service. In 273 inpatients, 46 (16.9%) of SABSIs were methicillin-resistant S. aureus (MRSA). MRSA was more frequent in those who had injected drugs in the past 6 months (20.6%) compared with other inpatients (15.7%). Injecting drug use was associated with a 4.82-fold (95% confidence interval = 1.54-16.29) increased odds of MRSA after accounting for confounders.

Multidrug tolerance conferred by loss-of-function mutations in anti-sigma factor RshA of Mycobacterium abscessus.

Low-level drug resistance in noncanonical pathways can constitute steppingstones toward acquisition of high-level on-target resistance mutations in the clinic. To capture these intermediate steps in Mycobacterium abscessus (Mab), we performed classic mutant selection experiments with moxifloxacin at twofold its minimum inhibitory concentration (MIC) on solid medium. We found that low-level resistance emerged reproducibly as loss-of-function mutations in RshA (MAB_3542c), an anti-sigma factor that negatively regulates activity of SigH, which orchestrates a response to oxidative stress in mycobacteria. Since oxidative stress is generated in response to many antibiotics, we went on to show that deletion of rshA confers low to moderate resistance-by measure of MIC-to a dozen agents recommended or evaluated for the treatment of Mab pulmonary infections. Interestingly, this moderate resistance was associated with a wide range of drug tolerance, up to 1,000-fold increased survival of a ΔrshA Mab mutant upon exposure to several β-lactams and DNA gyrase inhibitors. Consistent with the putative involvement of the SigH regulon, we showed that addition of the transcription inhibitor rifabutin (RBT) abrogated the high-tolerance phenotype of ΔrshA to representatives of the β-lactam and DNA gyrase inhibitor classes. In a survey of 10,000 whole Mab genome sequences, we identified several loss-of-function mutations in rshA as well as non-synonymous polymorphisms in two cysteine residues critical for interactions with SigH. Thus, the multidrug multiform resistance phenotype we have uncovered may not only constitute a step toward canonical resistance acquisition during treatment but also contribute directly to treatment failure.

Occurrence, Multidrug Resistance, and Multilocus Sequence Typing of Extended-Spectrum Beta-Lactamase/AmpC-Producing Escherichia coli from Farmed Eggs.

The present study evaluated the occurrence, antibiogram profile, and sequence types (STs) of multidrug resistant (MDR) Escherichia coli from freshly laid eggs (n = 480), feed (n = 24), water (n = 24), poultry droppings (n = 24), and hand swab samples (n = 10) collected from 24 deep litter (DL) and caged poultry layer farms (12 per category) across Punjab, India. The overall E. coli contamination rate in DL and cage farms was 32% (95% confidence intervals [CI], 26.6-37.8%) and 16.7% (95% CI, 12.6-21.6%), respectively. The logistic regression analysis revealed that the DL system had higher odds of occurrence (odds ratio [OR]) of extended-spectrum beta-lactamase (ESBL) (2.195, 95% CI, 1.065, 4.522) and ESBL/AmpC coproducers (2.69, 95% CI, 1.122, 6.45) compared to the cage system. Additionally, isolates from the DL were 4.065 (95% CI, 1.477, 11.188) times more tetracycline resistant compared to the latter; however, resistance to amoxyclavulanate (OR, 0.437; 95% CI, 0.209, 0.912), and ampicillin (OR, 0.343; 95% CI, 0.163, 0.720) was lesser in DL system. Notably, around 97.7% and 87.2% of the isolates from the DL and cage system were MDR, with the DL system having 6.439 (95% CI, 1.246, 33.283) times more chances of harboring MDR E. coli. Additionally, among the resistance genes, the DL system demonstrated significantly high presence of blaAmpC (56%), qnrA/B/S (42.3%), and tetA/B (30.6%). Furthermore, multilocus sequence typing of 11 MDR isolates (n = 5, DL, and 6, cage) revealed the presence of 10 STs, of which ST10, ST155, and ST156 were found to be of public health importance. Therefore, the present study highlights the burden of MDR, ESBL, and AmpC-producing E. coli on poultry eggs and farm environment, which could be carried over to human handlers and consumers upon direct contact during handling and processing.

Virulence, multiple drug resistance, and biofilm-formation in Salmonella species isolated from layer, broiler, and dual-purpose indigenous chickens.

Globally, the significant risk to food safety and public health posed by antimicrobial-resistant foodborne Salmonella pathogens is driven by the utilization of in-feed antibiotics, with variations in usage across poultry production systems. The current study investigated the occurrence of virulence, antimicrobial resistant profiles, and biofilm-forming potentials of Salmonella isolates sourced from different chicken types. A total of 75 cloacal faecal samples were collected using sterile swabs from layer, broiler, and indigenous chickens across 15 poultry farms (five farms per chicken type). The samples were analysed for the presence of Salmonella spp. using species-specific PCR analysis. Out of the 150 presumptive isolates, a large proportion (82; 55%) were confirmed as Salmonella species, comprising the serovars S. typhimurium (49%) and S. enteritidis (30%) while 21% were uncategorised. Based on phenotypic antibiotic susceptibility test, the Salmonella isolates were most often resistant to erythromycin (62%), tetracycline (59%), and trimethoprim (32%). The dominant multiple antibiotic resistance phenotypes were SXT-W-TE (16%), E-W-TE (10%), AML-E-TE (10%), E-SXT-W-TE (13%), and AMP-AML-E-SXT-W-TE (10%). Genotypic assessment of antibiotic resistance genes revealed that isolates harboured the ant (52%), tet (A) (46%), sui1 (13%), sui2 (14%), and tet (B) (9%) determinants. Major virulence genes comprising the invasion gene spiC, the SPI-3 encoded protein (misL) that is associated with the establishment of chronic infections and host specificity as well as the SPI-4 encoded orfL that facilitates adhesion, autotransportation and colonisation were detected in 26%, 16%, and 14% of the isolates respectively. There was no significant difference on the proportion of Salmonella species and the occurrence of virulence and antimicrobial resistance determinants among Salmonella isolates obtained from different chicken types. In addition, neither the chicken type nor incubation temperature influenced the potential of the Salmonella isolates to form biofilms, although a large proportion (62%) exhibited weak to strong biofilm-forming potentials. Moderate to high proportions of antimicrobial resistant pathogenic Salmonella serovars were detected in the study but these did not vary with poultry production systems.

Prevalence, Cross Contamination, Virulence, and Multidrug Resistance Profiles of Staphylococcus aureus Isolates from Four Middle-Scale Dairy Farms in Bareilly, Northern India.

Milk, a nutritious global important food commodity, serves as an excellent medium for microbial growth as well. The foodborne pathogen Staphylococcus aureus is a commensal member of human microflora that enters the food chain through poor hygienic practices and cross contamination and causes various clinical manifestations in humans. During this study, raw milk and swab samples (milker's hand, udder, towel, milking bucket, and farm floor) were collected from four middle-scale buffalo dairy farms. The results revealed S. aureus presence in 11.6% (n = 56/448) bucket raw milk samples and 2.6% (n = 12/448) udder raw milk samples. Contrarily, S. aureus prevalence was significantly higher in farm floors (100%, n = 84/84), towel (35.7%, n = 10/28), milking bucket (11.6%, n = 56/448), milker's hand (10.7%, n = 3/28), and udder swab samples (4.0%, n = 18/448). The chi-square test yielded p values of 0.000, 0.005, and 0.0011 for udder raw milk, udder swab, and milker's hand swab, respectively. The p values of the milking bucket (p = 0.048) and farm floors (p = 0.0183) confirmed their possible role in S. aureus cross contamination. Gene amplifications of nuclease and enterotoxin A indicate potential virulence of S. aureus isolates in collected samples. Antibiotic susceptibility testing revealed multidrug resistance in 44% (n = 239) of S. aureus isolates with the highest resistance of 61% against penicillin. Resistance against ampicillin, streptomycin, and lincomycin was observed. Fewer S. aureus isolates were resistant to kanamycin and erythromycin, whereas the lowest number of resistant isolates was observed against chloramphenicol. A high prevalence of S. aureus in the farm environment and milking equipment suggested the cross contamination of potentially enterotoxin-producing and multidrug-resistant S. aureus to raw milk. Therefore, good hygiene practices should be enforced to avoid foodborne and zoonotic infections.

Molecular surveillance of A. baumannii in intensive care units: exploration of transmission chains

Abstract Background Multidrug-resistant Acinetobacter baumannii (MDR-Ab) is one of the main causes of healthcare associated infections (HAIs). During the SARS-CoV-2 pandemic there was an increase in MDR-Ab infections, especially in intensive care units (ICUs). This study aimed to assess the potential spread or emergence of specific clusters of MDR-Ab across four different ICUs at the Umberto I teaching hospital of Rome. Methods From January 2020 to January 2022 microbiological surveillance was conducted in four ICUs: two dedicated to COVID-19 patients (ICU-1C, ICU-2C) and two to non-COVID-19 patients (ICU-1R, ICU-2R). The genetic relatedness between A. baumannii isolates was assessed using pulsed-field gel electrophoresis (PFGE). Illumina whole genome sequencing was conducted on 26 representative isolates. Results In total, 178 A. baumannii isolates were obtained from 129 COVID-19 patients and 49 non-COVID-19 patients. The isolates were classified into 17 PFGE pulsotypes, being two major (A, B) and five intermediate (C, D, E, H, Q). Clone A was present in all ICUs, while Clone B was present only in ICU-1C and ICU-2R. Overall, 117 isolates belonged to clone A and exhibited a MDR phenotype; all of them were placed within the international clonal lineage II. All isolates showed carbapenems resistance primarily attributed to the presence of the blaOXA-23 gene, while aminoglycosides resistance observed in almost all isolates was attributed to the presence of the armA gene. Small outbreaks involving intermediate pulsotypes were detected between ICU-1R and ICU-2R, ICU-1C and ICU-2R, ICU-1C and ICU-2C, ICU-1C and ICU-1R. Conclusions The observed outbreaks could be attributed to a decline in attention to normal care practices for the prevention of HAIs during the COVID-19 pandemic, favouring the spread of MDR-Ab. Therefore, it is recommended the strengthen of control measures and the implementation of long-term strategies targeting MDR microorganisms in the ICUs. Key messages • Acinetobacter baumannii was frequently found infecting patients across four different ICUs at the Umberto I teaching hospital of Rome. • Acinetobacter baumanni outbreaks and multidrug resistance were observed. Therefore, the implementation of long-term strategies targeting MDR microorganisms in the ICUs is highly recommended.

Restoring Colistin Sensitivity in Multidrug-Resistant Pathogenic E. coli Using Cinacalcet Hydrochloride

Restoring colistin’s efficacy is crucial in addressing the resistance crisis of colistin. This study utilized a high-throughput screening method to identify 43 compounds from 800 FDA-approved drugs that exhibited significant antibacterial effects when combined with colistin. Among these, cinacalcet hydrochloride (CH) was selected for its potential synergistic effect with colistin against multidrug-resistant (MDR) E. coli strains, including mcr-1-positive strains. A series of experiments revealed that the combination of CH and colistin showed strong synergy, especially in mcr-1-positive strains, restoring colistin sensitivity. The combination significantly inhibited bacterial growth and reduced CFU counts more effectively than either drug alone. Additionally, CH and colistin together significantly inhibited biofilm formation and eradicated existing biofilms, as visualized through confocal microscopy. Mechanistic studies showed that the combination increased bacterial membrane permeability and disrupted membrane integrity. The treatment also elevated extracellular ATP release and ROS production, indicating oxidative stress-induced bacterial death. Safety evaluations showed that the combination did not increase toxicity in host cells. Finally, animal models further validated the combination’s efficacy. Overall, this study showed that the combination of colistin and CH significantly restored colistin sensitivity in mcr-1-positive E. coli, revealing their synergistic antibacterial mechanism involving membrane damage and oxidative stress, with promising clinical applications.

The Challenge of Treating Infections Caused by Metallo-β-Lactamase-Producing Gram-Negative Bacteria: A Narrative Review.

Gram-negative multidrug-resistant (MDR) bacteria, including Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa, pose a significant challenge in clinical practice. Infections caused by metallo-β-lactamase (MBL)-producing Gram-negative organisms, in particular, require careful consideration due to their complexity and varied prevalence, given that the microbiological diagnosis of these pathogens is intricate and compounded by challenges in assessing the efficacy of anti-MBL antimicrobials. We discuss both established and new approaches in the treatment of MBL-producing Gram-negative infections, focusing on 3 strategies: colistin; the recently approved combination of aztreonam with avibactam (or with ceftazidime/avibactam); and cefiderocol. Despite its significant activity against various Gram-negative pathogens, the efficacy of colistin is limited by resistance mechanisms, while nephrotoxicity and acute renal injury call for careful dosing and monitoring in clinical practice. Aztreonam combined with avibactam (or with avibactam/ceftazidime if aztreonam plus avibactam is not available) exhibits potent activity against MBL-producing Gram-negative pathogens. Cefiderocol in monotherapy is effective against a wide range of multidrug-resistant organisms, including MBL producers, and favorable clinical outcomes have been observed in various clinical trials and case series. After examining scientific evidence in the management of infections caused by MBL-producing Gram-negative bacteria, we have developed a comprehensive clinical algorithm to guide therapeutic decision making. We recommend reserving colistin as a last-resort option for MDR Gram-negative infections. Cefiderocol and aztreonam/avibactam represent favorable options against MBL-producing pathogens. In the case of P. aeruginosa with MBL-producing enzymes and with difficult-to-treat resistance, cefiderocol is the preferred option. Further research is needed to optimize treatment strategies and minimize resistance.

Antimicrobial stewardship program in one primary care hospital, a snapshot analysis

Abstract Antimicrobic resistance (AMR) continues to rise, whereas development of new agents to counter it has slowed. A heightened need to maintain the effectiveness of currently available agents exists. Healthcare-associated infections (HAIs) are a significant cause of poor treatment outcomes and elevated healthcare and societal costs worldwide. At the beginning of 2024, we started a prospective, interventional, interrupted time-series study, based on Prospective Audit and Feedback in the Intensive Care Unit (ICU) and in the internal medicine ward of our hospital. The primary outcomes will be the difference in the antibiotic consumption, and the incidence of bloodstream infections (BSI) caused by multidrug-resistant (MDR) organisms. The secondary outcomes will include the hospital mortality rate, the mean length of stay and the antibiotic expense. We conducted a snapshot analysis of the first three months to assess the efficacy our program. In the first three months we registered a global reduction in antibiotic expense (almost by half in comparison of 2023 first quarter) and consumption, particularly in the use of carbapenems: (change in level, CL: -51,26 DDD/1000 PD,) and third- generation cephalosporins (CL: -6 DDD/1000 PD). We registered a reduction in the use of hydroalcoholic gel, probably due to the reducted perception after the waning of risk perception towards Covid-19, although no big difference was observed in BSI incidence due to MDR Gram-negative organisms (CL: +1,2 events/1000 PD). Albeit partial, once again these findings demonstrated that implementation of an antimicrobial stewardship program, no matter the level of healthcare, is critical for fighting the AMR and to lessen the burden on Public Health; in particular our program induced a significant reduction in antibiotic consumption that lead to more funds at disposal to improve our clinical practice. Key messages • Once again our findings demonstrated that implementation of an antimicrobial stewardship, no matter the level of healthcare, is critical for fighting the AMR. • Our program induced a significant reduction in antibiotic consumption.

Rapid increase of multi-drug resistant Pseudomonas aeruginosa in Greece - WHONET-Greece (2020-2023)

Abstract Background Multidrug-resistant (MDR) Pseudomonas aeruginosa has been documented as a significant threat associated with adverse patient outcomes. We aimed to describe the rapid increase in the prevalence of an MDR P.aeruginosa phenotype at the national level, as detected through the Greek Electronic System for the Surveillance of Antimicrobial Resistance (WHONET-Greece). Methods Routine susceptibility data of 22,236 P.aeruginosa isolates of hospitalized patients in forty-five hospitals, isolated over the past four years (2020-2023) were analyzed using ‘Resistance profiles’ analysis of WHONET software(ver.2023), considering five antimicrobial groups/agents. Results We confirmed the rapid increase in the prevalence of a carbapenem-resistant P.aeruginosa (CRPA) phenotype resistant to all five antimicrobial groups/agents tested (carbapenems, piperacillin-tazobactam, cephalosporins, fluoroquinolones and aminoglycosides). The prevalence increased by 63.8% in 2023 (from 15.2% to 24.9%, 1.64-fold increase), whereas in the last 24 months of the study period a 2.13-fold increase was observed (from 11.7% to 24.9%). In parallel, the prevalence of all other CRPA profiles decreased in 2023 from 22.5% to 17%. We also examined the prevalence of this MDR phenotype in the different NUTS-1 regions of the country, which revealed that the rapid increase is mainly observed in Attika (EL3, 19/45 hospitals) and Central Greece (EL6, 8/45 hospitals) where prevalence reached 25.4% and 26.2% in the second half of 2023 respectively. Northern Greece (EL5, 15/45 hospitals) showed a steady increase over the four years (12% to 19.3%), while in the Aegean Islands/Crete region (EL4, 3/45 hospitals) the prevalence levels were stable. Conclusions There is an emerging multi-drug CRPA phenotype in Greece. Only with systematic, nationwide surveillance could we identify and verify such threats. The findings of this study should be used for targeted interventions and further analysis through molecular techniques. Key messages • The early detection of such emerging multi-drug CRPA phenotype in Greece allows for for targeted interventions and further analysis through molecular techniques. • Systematic surveillance of antimicrobial resistance based on routine susceptibility data is a great way to identify and verify emerging public health threats.