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The aim of this study was to understand the molecular epidemiological characteristics and drug resistance mechanism of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and to provide a theoretical basis for the prevention and treatment of CRPA infection in hospitals. A total of 34 CRPA strains were isolated, and resistance to 13 commonly used antibiotics was detected using the TDR-300B Plus VitEK-2 compact automatic bacterial identification instrument. Then, carbapenemase production was detected using the Carbe NP test. RT-qPCR was used to detect the expression of efflux pump MexA and outer membrane protein OprD, and PCR amplification and sequence analysis were used to detect class Ⅰ integrons carried by drug resistance genes. Our results showed that of the 34 CRPAs, 22 were multi-drug resistant (MDR), and five were extensively drug-resistant (XDR). Sequencing analysis showed that class Ⅰ integron mainly carried aminoglycosides or quinolones resistance genes. Multiple mechanisms play important roles in the formation and development of MDR or XDR.

The rise of multidrug resistance (MDR) bacteria in nosocomial and health-care institutions is widespread and is currently recognized as a major medical challenge. Mechanisms of bacterial resistance, namely, quorum sensing (QS), biofilm formation, and efflux pumps, have been identified as critical biological processes in MDR bacteria. Following previous reports on the activity of phenothiazines against mechanisms of bacterial resistance, in this work we focus on the synthesis of xanthene derivatives aiming to discover phenothiazine bioisosteres with improved activity. Four compounds were obtained from the conjugation of xanthydrol with sulfonamides and aniline and were fully characterized. Their antibacterial activity was assessed considering their minimum inhibitory concentration (MIC) against Gram-positive and Gram-negative strains, efflux pump inhibition, influence on biofilm formation and quorum-sensing (QS) inhibition. It was observed that the MIC of all the tested compounds was above 64 µg/mL The four 9-xanthenyl derivatives obtained, particularly the xanthene sulfonamide derivatives 3b and 3c, showed promising results on QS inhibition with a reduction of pigment production of 48 and 41 mm, and on biofilm formation with a reduction of 78 and 79%, respectively.

Background:The emergence and spread of extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae is now a major challenge in treating urinary tract infections. This study was conducted to determine the Prevalence of ESBL genes inKlebsiella pneumoniaeand the influence of these genes on the antimicrobial susceptibility patterns of the isolates, obtained from community acquired urinary tract infection in Enugu state rural communities. Method:A total of 735 clean catch mid-stream urine samples were collected from February, 2021 to June, 2021. The urine samples were cultured andKlebsiella pneumoniaewere identified morphologically, biochemically, and then typed down, using PCR, Gel electrophoresis, and Sanger Sequencing by Genewiz. Antibiotic susceptibility testing was done by modified Kirby-Bauer disc diffusion method and interpretation was done following Clinical and Laboratory Standard Institute (CLSI) guidelines. ESBL screening was done by the phenotypic method, using the standard disk diffusion method, whereas the phenotypic confirmation of ESBL producers was done by the double-disk synergy method, and interpreted according to CLSI guidelines. Multiplex PCR was used to detect the genes for SHV and CTX-M while conventional linear PCR was done for TEM andblasGES type ESBL genes. Results:A total of 77 isolates were identified asKlebsiella pneumoniae, and the prevalence of positive ESBL in them were 10(12.9%). Of the 77 isolates that were tested for antibiogram, 29 isolates were multidrug-resistance (MDR). Out of the MDR isolates, 10 were ESBL positive, whereas 19 were not. Of this 29 MDR isolates, 6 were extensively-drug resistant (XDR). Of the 6 XDR isolates, 3 do not possess ESBL enzymes, whereas 3 of them do. Age range of 31-40 contributed the highest prevalence of ESBL genes (60%), whereas age range of 10-20, 21-30, and 51-70 did not produce any ESBL gene.blaTEM gene was the most prevalent ESBL resistant genes with a prevalence of 10 (100%), followed by SHV 9(90%), whereasblaGES gene andblaCTX-M have the least prevalence of 4 (40%) each. Conclusion:Higher prevalence of MDR, XDR, and ESBL-producingKlebsiella pneumoniaewere observed, thus the need for public health intervention for effective prevention and control of antimicrobial resistance, and proper treatment of UTIs.

Executive Summary The present Deliverable, ‘D9.2 – In-depth analysis of legal and ethical requirements’, part of Work Package 9 ‘Ethical and Legal Framework’ (WP9) of the In Silico World project, assesses the core pieces of legislation and ethical principles identified in Deliverable ‘D9.1 – Legal and Ethical Inventory’. The report analyses the following areas of legislation – and identifies some key issues – relevant to the In Silico World project and, thus, for in silico trials: Privacy and Data Protection: Data protection is a long-established legislation in the European Union (EU), which has evolved throughout the last decades, and the GDPR is the fundamental EU law to consider for in silico trials. Hence, the report introduces the principles of data processing, the notion of personal data and health data and the legal bases for data processing. In silico trials may highlight some of the common challenges for the healthcare sector in data protection. These include the legal bases and the further processing of health data. In silico trials may also raise interpretative questions. These include doctrinal discussions about the nature of synthetic data in their relationship with anonymization and pseudonymisation, and the concept of data ownership. Data Governance: The legal landscape concerning health data sharing is changing due to a series of new EU legislative initiatives. These initiatives encompass the Data Governance Act, the European Health Data Space (EHDS) proposal, and the Data Act proposal. The Data Governance Act sets rules for re-using certain categories of personal data and introduces the concept of data altruism in healthcare. The EHDS proposal regulates, inter alia, the primary and secondary use of health data. The Data Act proposal proposes business-to-consumer, business-to-business and business-to-government data sharing rules. These three pieces of legislation are expected to apply simultaneously once all are approved. Nevertheless, some challenges may arise in the future, which may concern the appropriate legal basis for data sharing and data altruism, and the interaction of these with the GDPR and national legislation. Clinical Trials, Medicinal Products and Medical Devices: Clinical trials, medicinal products and medical device legislation are relevant to the very essence of in silico trials. For clinical trials and medicinal products, Regulation 726/2004 provides the legal basis for the EMA to deal with novel methodologies for drug development. The Medical Device Regulation (MDR) and In Vitro Medical Device Regulation (IVDR) are the applicable laws for medical device legislation and explicitly mention modelling and simulation. However, while these laws do not prohibit in silico trials, they do not extensively address them either. There exist challenges and barriers that need to be addressed both on a regulatory and legislative perspective. On the regulatory side, guidance and standardisation efforts are needed in general (to tackle, for example, Artificial Intelligence (AI)) and in particular (on the verification and validation of in silico models). On the legislative side, there is common agreement that the current pharmaceutical legal framework lacks behind digital innovation processes. The EU Pharmaceutical Strategy promises the reform of the existing pharmaceutical framework. It is desirable that the reform will address more comprehensively the innovative aspects of the medicinal product’s lifecycle, including in silico trials. Artificial Intelligence: The legal framework of AI is in the process of being established in the EU. The AI Act proposal, expected to be approved in 2023, will introduce new requirements for providers, users and all the actors involved in AI systems. These include, inter alia, adopting a risk management system, a quality management system, documentation duties, ensuring transparency, human oversight, accuracy, robustness, and cybersecurity. The AI Act will be relevant for medical devices and in vitro diagnostic medical devices, as – according to the latest available version of the proposal – they are explicitly included in the scope of the regulation. The regulation sparked several discussions that this report cannot summarise comprehensively. Therefore, the report chooses a new item currently negotiated in the latest proposal’s version may be of crucial relevance for medicine and healthcare: the potential non-application of AI rules in the context of scientific research. The preliminary conclusion is that these rules, as currently formulated, may generate legal uncertainties in the future. The report concludes with a section on the ‘Ethics Principles’. The section is based on the Biomedical Ethics principles that were illustrated in D9.1, i.e. autonomy, justice, beneficence, and non-maleficence. 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With the deadline to reach net-zero quickly approaching, the aviation industry will soon have to shift away from kerosene aircraft. One of the candidate zero-emissions technologies for future aircraft is hydrogen. However, due to the technology being yet immature, the implementation of hydrogen on aircraft is expected to be a long-term endeavour. To accelerate this process, a study has been conducted to evaluate the feasibility of hydrogen-kerosene hybrid combustion as a stepping stone towards full hydrogen implementation. Three goals have been defined: to estimate first-order performance of future hydrogen combustion aircraft, to bring understanding to the hydrogen combustion design space, and to identify a potential pathway for the implementation of hydrogen combustion. To achieve these, simulations have been run using a computational model of a hydrogen retrofit of a ATR 72-500 with engines capable of hybrid combustion. An optimization approach was used to explore this new design space without biases. To accurately reflect the importance of each optimization objective, Tabu Search with Multiple Dominance Relations was chosen as the optimizer. The results of this investigation have revealed fundamental trade-offs in the retrofit design space and provided a set of optimized aircraft of varying hybridisation fraction that can pave the pathway towards full hydrogen implementation in aircraft.

Highlights • Different fishes were collected and were subjected to form an in vitro biofilm. • Huge array (up to 107 cfu/ml or g) of pathogenic bacteria. • Few of the isolates were sensitive and few were resistant against the antibiotics but after bio-film formation all the species acquired resistance., Background The ability of many bacteria to adhere on the host surfaces and forming biofilms has major implications in a wide variety of industries including the food industry, where biofilms may create a persistent source of contamination. In the same environmental condition, the multiple bacterial species can closely interact with each other and may easily enhance their drug resistance capability, which finally increases the multi-drug resistant (MDR) attribute of the species. Objective The present study examined whether the mixed-species biofilm possesses any impact on the enhancement of the antibiotic resistance of the planktonic or single-cell bacterial isolates present in the fish samples. Methods In this regard, Cyprinus rubrofuscus (Koi), Heteropneustes fossilis (Shing) and Mystus vittatus (Tengra) fishes were collected and subjected to form an in vitro biofilm by shaking condition into the wise bath. The drug-resistant pattern was determined by the Kirby Bauer technique. Results All the samples exhibited a huge array (up to 107 cfu/ml or g) of bacteria such as E. coli, Klebsiella spp., Vibrio spp., Salmonella spp., Proteus spp. and Staphylococcus spp. The isolates from both the bulk samples and their corresponding biofilms were subjected to antibiogram assay using antibiotics such as Ampicillin (10 µg), Erythromycin (15 μg), Streptomycin (STP 10 μg), Oxacillin (10 µg), Nalidixic acid (30 µg). Before biofilm formation, few of the isolates were found to be sensitive and few were resistant against the antibiotics. But when the species were isolated from the biofilm the sensitive one acquired drug resistance and resistant strain unveiled more resistance towards the same antibiotics. The present study revealed extensive bacterial contamination in fish samples among those some were resistant against the supplied drugs. Conclusion After the formation of multi-species biofilm, the isolates became more resistant against the same drugs that is alarming for consumers and major obstacles to maintain sustainable health.

The treatment of K. pneumoniae infections has become more difficult due to the emergence of multidrug-resistant strains. This study aimed to evaluate antimicrobial resistance patterns and detect different types of beta-lactamase genes among MDR and non-MDR Klebsiella pneumoniae in various clinical samples. A total of 150 Klebsiella pneumoniae were identified from different clinical samples by conventional microbiological procedures. Antibiotic sensitivity was detected by the Kirby-Bauer disc diffusion method. Extended-spectrum beta-lactamase, Metallo-β-lactamase, and carbapenemase were phenotypically detected by double-disk synergy test, combined disk assay, and modified Hodge test. PCR also detected different β lactamase genes and virulence genes. The majority of Klebsiella pneumoniae were multi-drug resistant (82%). The bacteria were found resistant to most β-lactam antibiotics, aminoglycosides, ciprofloxacin, cotrimoxazole, carbapenem, piperacillin, and tazobactam. However, only about 0.7% colistin resistance was observed. A double-disk synergy test was performed among the isolates for phenotypic detection of ESBL producing Klebsiella pneumoniae. Only 16.6% of isolates were found to be ESBL producers. Among β-lactamase genes, NDM (23.34%), OXA-48(8%), and KPC (7.3%) were detected. Other prevalent β lactamase genes VIM and IMP were not detected. In summary, the prevalence of MDR Klebsiella pneumoniae is high in Bangladesh, which may complicate the treatment of hospitalized patients.

The development of carbapenem resistance in extraintestinal pathogenic Escherichia coli (ExPEC) has significant clinical implications, particularly in countries where second-line antimicrobials are not readily available, rendering treatments ineffective, and ExPEC infections untreatable. Thus, early detection of high-risk ExPEC lineages and raising awareness of the specific mechanisms underlying carbapenem resistance are mandatory for the selection of appropriate treatment options and the prevention of E. coli spread. This study aims to investigate the phenotypic and genotypic features of the first NDM-5 carbapenemase-producing ExPEC strain isolated from the blood of a patient admitted to the Maputo Central Hospital (MCH), in Mozambique. E. coli SSM100 isolate was identified by MALDI-TOF, it displayed high-level resistance to third generation cephalosporins, carbapenems, fluoroquinolones, and aminoglycosides, performing antimicrobial susceptibilities testing by VITEK 2 system. E. coli SSM100 isolate was classified through whole-genome sequencing as ST405-D-O102: H6, a globally distributed lineage associated with antimicrobial resistance, carrying the blaNDM-5 gene located on an F1:A1:B49 plasmid, coharboring blaCTX-M-15, blaTEM-1, aadA2, sul1, and dfrA12 genes. In addition, mutations in gyrA (S83L and D87N), parC (S80I and E84V), and parE (I529L) conferring fluoroquinolone resistance were also found. Moreover, SSM100 isolate carried 88 virulence genes, of which 28 are reported to be associated with UPEC. The emergence of NDM-5 carbapenemase in a pandemic ST405-D-O102:H6 clone in Mozambique is of great concern. Locations of extended-spectrum β-lactamase determinants and NDM-5 carbapenemase gene on IncF-plasmid can increase their spread reinforcing the need for antimicrobial surveillance and the urgent introduction of carbapenemase detection tests in diagnostic laboratories of the country.

Product development in medical technology is largely governed by Regulatory Affairs. In contrast, for consumer products Human-Centred Design (HCD) is the guiding principle. Both approaches are laid down in international standards and overlap many aspects such as procedure, applied methodologies and development activities. For medical devices patient safety and therefore risk management is the guiding development goal. In HCD the focus lies on User Experience (UX), a positive experience throughout the entire product life cycle. The question arises whether UX could play a more important role in medical device development, particularly considering the similarities of the two development processes

Klebsiella species is commonly associated with serious nosocomial infections. Multi-drug resistant Klebsiella species isolates are becoming increasingly prevalent in the clinical and nosocomial environments. The high prevalence of Klebsiella infections is related to the ability of Klebsiella species to acquire and disseminate exogenous genes associated with mobile elements, such as plasmids, transposons and integrons. This study was conducted to find out the presence of class 1 integron and antibiotic susceptibility patterns of Klebsiella species from clinical specimens at No (1) Defence Services General Hospital (1000-Bedded). A laboratory based cross-sectional descriptive study was carried out from January to september, 2020. Identification and antimicrobial susceptibility testing of Klebsiella species was performed by VITEK 2 Compact Analyzer. Class 1 integrons were detected by conventional PCR. In this study, 110 Klebsiella species were isolated from various clinical specimens and most of the isolates were from medical ward (52.3%, 63 isolates). The highest rate of resistance was observed for ampicillin (100%) and Cefotaxime (97.3 %) whereas the lowest antibiotic resistance was to Amikacin (15.5%). Out of 110 Klebsiella isolates, 107 (97.3%) were multidrug resistant (MDR). Forty-four (40%) out of 110 Klebsiella isolates carried int1 gene and all these isolates were MDR. However, there was no association between multidrug resistance and integron positivity (p value = 0.273). The presence of class I integron genes among Klebsiella species highlights the continued monitoring is necessary for prevention of wide dissemination of integrons and infections by MDR pathogens.

Background: The noteworthy spread of carbapenem-resistant K. pneumoniae (CR-KP) isolates represents a significant safety threat. Objective: Determination of the carbapenemase genes incidence among CR-KP clinical isolates in Kafrelsheikh, Egypt. Methods: A total of 230 K. pneumoniae isolates were recovered from four hospitals in Kafrelsheikh, Egypt. Susceptibility testing was conducted using Kirby-Bauer method and automated-Vitek2 system. CR-KP isolates were tested using modified Hodge test (MHT) and combined disk synergy test. PCR and DNA sequencing were conducted for CR-KP isolates to rec- ognize the included carbapenemase-genes. Results: Out of 230 K. pneumoniae isolates, 50 isolates presented resistance to carbapenem (meropenem). All 50 CR-KP iso- lates were multidrug-resistant (MDR). Genes like blaNDM-1 and blaOXA-48 were the only detected genes among CR-KP with an incidence of 70.0% and 52.0%, respectively. Up to 74.0% of the tested isolates carried at least one of the two record- ed genes, among them 48.0% co-harbored both blaNDM-1 and blaOXA-48 genes. The accession-numbers of sequenced blaNDM-1 and blaOXA-48 genes were MG594615 and MG594616, respectively. Conclusion: This study reported a high incidence of MDR profile with the emergence of blaNDM-1 and blaOXA-48 genes co-existence in CR-KP isolates in Kafrelsheikh, Egypt. Hence, more restrictions should be applied against the spread of such serious pathogens. Keywords: Klebsiella pneumoniae; Egypt; carbapenem resistance; MDR; PCR; blaNDM-1; blaOXA-48; sequencing.

Met de komst van Verordening (EU) 2017/745 van het Europees Parlement en de Raad van 5 april betreffende medische hulpmiddelen, tot wijziging van Richtlijn 2001/83/EG, Verordening (EG) nr. 178/2002 en Verordening (EG) nr. 1223/2009, en tot intrekking van Richtlijnen 90/385/EEG en 93/42/EEG van de Raad, beter bekend als de Medical Device Regulation (MDR), kwam er ook meer aandacht voor goede klinische praktijken en de internationale norm ISO 14155 (Klinisch onderzoek van medische hulpmiddelen voor gebruik bij mensen — Goede klinische praktijkrichtlijnen (GCP)). Op zich is er niets verkeerd aan méér aandacht voor goede klinische praktijken, maar in combinatie met de MDR levert dat ook een paar misverstanden op. Een van die misverstanden betreft de veronderstelling dat onder de MDR al het klinisch onderzoek met medische hulpmiddelen in overeenstemming met goede klinische praktijken uitgevoerd moet worden. In deze blog post zet ik uiteen waarom dat alleen geldt voor klinisch onderzoek met medische hulpmiddelen dat valt onder MDR artikel 62 of 74 en niet voor het overig klinisch onderzoek dat onder artikel 82 valt.

Executive Summary: The present Deliverable ‘D9.1 – Legal and Ethical Inventory’, part of Work Package 9 ‘Ethical and Legal Framework’ of the In Silico World project, provides an overview of the core pieces of legislation and ethical principles that are relevant to the In Silico project, and thus, in silico trials. The analysis finds the following areas of legislation are of relevance: Privacy and Data Protection: The data processed for in silico trials entail the processing of personal data of individuals (notably, patients) in healthcare settings. As a consequence, privacy and data protection legislation becomes relevant. Privacy and data protection are distinct fundamental rights enshrined in the European Union’s (EU) treaties. The core and directly applicable regulation concerning the processing of personal data is the General Data Protection Legislation (GDPR). Other important data protection initiatives are undertaken by the Council of Europe and the Organisation for Economic Co-operation and Development (OECD). Data Governance: In healthcare and in silico trials, the sharing of data across healthcare facilities may be considered a key factor for the development, testing, and validation of in silico models. The European legislator’s and national Member States' growing interest in regulating data sharing has resulted in a new and rapidly evolving framework concerning the governance of data. The current EU legal framework on data governance includes the Open Data Directive, the Free Flow of Non-Personal Data Regulation, and the Data Governance Act. In addition to these, two legislative proposals are expected to radically change data sharing in the healthcare sector: the Data Act and the European Health Data Space. Clinical Trials: The legislation and regulatory documentation concerning clinical trials are relevant to the very essence of in silico trials. One of the main barriers envisaged by the In Silico World project is the lack of clear regulatory pathways for in silico trials. Illustrating the clinical trials' fundamental pieces of legislation in Europe is crucial for the project's objectives. The most relevant items of clinical trials legislation in the EU include the Declaration of Helsinki, the Declaration of Taipei, the Clinical Trials Directive, the Good Clinical Practice Directive, and the Clinical Trials Regulation. Medical Devices and Medicinal Products: From a product regulation perspective, in silico trials encompass medical devices and medicinal products. Concerning the first, the Medical Device Regulation and the In Vitro Medical Device Regulation are of primary importance, unitedly with the EU-level relevant bodies' guidance issued by the Medical Device Coordination Group. Concerning medicinal products, the analysis includes the Community Code for Medicinal Products for Human Use Directive 2009/94, the Advanced Therapeutic Medicinal Product Regulation, and the Health Technology Assessment Regulation. Artificial Intelligence (AI): It is no surprise that a fundamental component of in silico trials is artificial intelligence. The EU legal framework on Artificial Intelligence is in the process of being established. After the White Paper on Artificial Intelligence and the Guidelines on Trustworthy elaborated by the EU High-Level Expert Group on AI, the piece to monitor in the following months is the Artificial Intelligence Act. This proposal, if approved, will apply directly to all the EU Member States, and it will include general provisions of relevance to healthcare (including, for instance, medical devices) and in silico trials. In addition to the above pieces of legislation, the study outlines the framework for forthcoming Ethics guidelines. Based on a principlist approach, these guidelines propose the analysis of four main Biomedical Ethics principles – i.e. autonomy, justice, beneficence, non-maleficence – and the secondary principle of responsibility. These principles will ultimately be beneficial for studying the fundamental ethical principles that may be used as guidance for interpreting the law and supporting stakeholders to ensure the protection and advancement of human values in the context of in silico trials. The deliverable provides a descriptive overview of the above mentioned legal frameworks. It is prodromic to the next phases of the WP9, where the analysis will adopt an evaluative approach on legislation (D9.2, D9.3) to ultimately result in policy recommendations (D9.4).

Ventilator-associated pneumonia (VAP) is associated with increased hospital stay and high morbidity and mortality in critically ill patients. The aims of this study were to (i) determine the incidence of multidrug-resistant (MDR) pathogens in the first episodes of VAP and to assess potential differences in bacterial profiles of subjects with early- versus late-onset VAP. This was a retrospective cohort study over a period of 18 months including all patients who had a first episode of VAP confirmed by positive bacterial culture. Subjects were distributed into two groups according to the number of intubation days: early-onset VAP (&lt, 5 days) or late-onset VAP (≥5 days). The primary endpoint was the nature of causative pathogens and their resistance profiles. Sixty patients were included, 29 men and 31 women, with an average age of 38 ± 16 years. The IGS 2 at admission was 40.5 [32–44] and APACHE was 19 [15–22]. Monomicrobial infections were diagnosed in 77% of patients (n = 46). The most frequently isolated bacteria were A. baumannii, 53% (n = 32), P. aeruginosa in 37% (n = 22), Enterobacterales in 28% (n = 17) and S. aureus in 5% (n = 3). Ninety-seven percent of the bacteria were MDR. The VAP group comprised 36 (60%) episodes of early-onset VAP and 24 (40%) episodes of late-onset VAP. There was no significant difference in the distribution of the bacterial isolates, nor in terms of antibacterial resistances between early- and late-onset VAPs. Our data support recent observations that there is no microbiological difference in the prevalence of potential MDR pathogens or in their resistance profiles associated with early- versus late-onset VAPs, especially in countries with high rates of MDR bacteria.

Liang Du,1 Yu Zhang,1 Xintong Lv,2 Yuxin Duan,1 Xiaoyan Shi,2 Haoqiang Ji,1 Ruiheng Wu,1 Jia Xu,1 Xu Chen,1 Yang Gao,2 Xiwei Lu,2 Ling Zhou1 1School of Public Health, Dalian Medical University, Dalian, Liaoning, 116044, People’s Republic of China; 2Dalian Tuberculosis Hospital, Dalian, Liaoning, 116031, People’s Republic of ChinaCorrespondence: Xiwei Lu; Ling Zhou 9 West Section, Lvshun South Road, Dalian, Liaoning Province, People’s Republic of ChinaTel +86 411 8611 0368Email tb_luxiwei@126.com; zhouling0609@163.comPurpose: Multidrug-resistant tuberculosis (MDR-TB) is the cause of serious health and economic burdens worldwide. The present study aimed to explore the initial and acquired drug-resistance rates among TB patients from 2012 to 2019 in Dalian, China. The effectiveness of MDR-TB prevention and control strategies were then evaluated.Patients and Methods: Drug susceptibility testing (DST) was performed for 6429 diagnosed, culture-positive, Mycobacterium tuberculosis (MTB) strains, including 4661 new cases and 1768 previously treated cases. Descriptive statistics were employed to calculate the frequencies and percentages of TB strains, and the average annual growth rates (AAGRs) for each strain were calculated. The Chi-square test was applied to examine the significance of linear drug-resistance trends over time during the study period.Results: Over the eight-year study period, the percentages of both initial (from 9.01% to 4.82%) and acquired (from 40.85% to 9.09%) MDR-TB cases decreased significantly, AAGRs of 8.55% and 19.32%, respectively. Among new and previously treated TB patients, significant downtrends were observed for the rates of both initial and acquired MDR-TB among young and middle-aged individuals (P < 0.05). Additionally, among both new and previously treated TB patients, the percentages of individuals with drug resistance against isoniazid (INH), rifampicin (RFP), ofloxacin (OFX), and amikacin (AMK) decreased significantly (P < 0.05) from 2012 to 2019 in Dalian, China.Conclusion: The initial and acquired multidrug resistance rates exhibited significantly decreasing trends from 2012 to 2019, suggesting that MDR-TB prevalence has been controlled effectively in Dalian, China. The MDR-TB epidemic was reversed in the short term by establishing feasible strategies for detection, diagnosis, treatment, and infection control.Keywords: TB, initial, acquired, MDR, epidemic

Klebsiella pneumoniae, a member of the Enterobacteriaceae family, has become a dangerous pathogen accountable for a large fraction of the various infectious diseases in both clinical and community settings. In general, the K. pneumoniae population has been divided into the so-called classical (cKp) and hypervirulent (hvKp) lineages. The former, usually developing in hospitals, can rapidly acquire resistance to a wide spectrum of antimicrobial drugs, while the latter is associated with more aggressive but less resistant infections, mostly in healthy humans. However, a growing number of reports in the last decade have confirmed the convergence of these two distinct lineages into superpathogen clones possessing the properties of both, and thus imposing a significant threat to public health worldwide. This process is associated with horizontal gene transfer, in which plasmid conjugation plays a very important role. Therefore, the investigation of plasmid structures and the ways plasmids spread within and between bacterial species will provide benefits in developing prevention measures against these powerful pathogens. In this work, we investigated clinical multidrug-resistant K. pneumoniae isolates using long- and short-read whole-genome sequencing, which allowed us to reveal fusion IncHI1B/IncFIB plasmids in ST512 isolates capable of simultaneously carrying hypervirulence (iucABCD, iutA, prmpA, peg-344) and resistance determinants (armA, blaNDM-1 and others), and to obtain insights into their formation and transmission mechanisms. Comprehensive phenotypic, genotypic and phylogenetic analysis of the isolates, as well as of their plasmid repertoire, was performed. The data obtained will facilitate epidemiological surveillance of high-risk K. pneumoniae clones and the development of prevention strategies against them.

Global dispersion, hospital outbreaks, and lineage relationships between emerging antibiotic-resistant strains such as Klebsiella pneumoniae are of public health interest. This study aimed to isolate and identify K. pneumoniae clones from third-level healthcare hospitals in Mexico to establish their multidrug-resistant phenotype, phylogeny, and prevalence. Biological and abiotic surface samples were used to isolate K. pneumoniae strains and to test their antibiotic susceptibility to classify them. The housekeeping genes: gapA, InfB, mdh, pgi, phoE, ropB, and tonB were used for multilocus sequence typing (MLST). Phylogenetic networks were constructed with 48 strains. Isolated strains (93) were mainly from urine and blood, 96% were resistant to ampicillin as expected, 60% were extended-spectrum β-lactamases (ESBL), 98% were susceptible to ertapenem and meropenem and 99% were susceptible to imipenem, 46% were multi-drug resistant (MDR), 17% were extensively-drug resistant (XDR), 1% were pan-drug resistant (PDR), and 36% were not classified. The tonB, mdh, and phoE genes were the most variable, and the InfB gene showed positive selection. The most prevalent sequence types (STs) were ST551 (six clones), ST405 (six clones), ST1088 (four clones), ST25 (four clones), ST392 (three clones), and ST36 (two clones). ST706 was PDR, and ST1088 clones were MDR; neither of these STs has been reported in Mexico. The strains analyzed were from different hospitals and locations; thus, it is important to maintain antibiotic surveillance and avoid clone dissemination to prevent outbreaks, adaptation to antibiotics, and the transmission of antibiotic resistance.

A new series of nitric oxide-releasing estra-1,3,5,16-tetraene analogs (NO-∆-16-CIEAs) was designed and synthesized as dual inhibitors for EGFR and MRP2 based on our previous findings on estra-1,3,5-triene analog NO-CIEA 17 against both HepG2 and HepG2-R cell lines. Among the target compounds, 14a (R-isomer) and 14b (S-isomer) displayed potent anti-proliferative activity against both HepG2 and HepG2-R cell lines in comparison to the reference drug erlotinib. Remarkably, compound 14a resulted in a prominent reduction in EGFR phosphorylation at a concentration of 1.20 µM with slight activity on the phosphorylation of MEK1/2 and ERK1/2. It also inhibits MRP2 expression in a dose-dependent manner with 24% inhibition and arrested the cells in the S phase of the cell cycle. Interestingly, compound 14a (estratetraene core) exhibited a twofold increase in anti-proliferative activity against both HepG2 and HepG2-R in comparison with the lead estratriene analog, demonstrating the significance of the designed ∆-16 unsaturation. The results shed a light on compound 14a and support further investigations to combat multidrug resistance in chemotherapy of hepatocellular carcinoma patients.

Objectives: Brucella anthropi is a Gram-negative, aerobic, motile, oxidase-positive, non-fermentative Alphaproteobacteria belonging to the family Brucellaceae. It is most commonly found in soil but is an emerging, opportunistic, nosocomial human pathogen. The objective of this study was to understand the genome features of a drug-resistant B. anthropi (SOA01) isolated from a blood culture of a 4-day-old neonate and to determine its antimicrobial resistance and pathogenic potential. Methods: Hybrid genome assembly of B. anthropi strain SOA01 was generated using quality-trimmed short Illumina and long MinION reads. Identification and antimicrobial susceptibility profile were determined by MALDI-TOF, in silico ribosomal multilocus sequence typing (rMLST) and VITEK®2, respectively. PATRIC webserver and VFDB were used to identify antimicrobial resistance (AMR), virulence factor (VF) and transporter genes. Results: Multidrug-resistant B. anthropi strain SOA01 has a genome of 4 975 830 bp with a G+C content of 56.29. Several AMR, VF and transporter genes were identified in the genome. Antimicrobial susceptibility testing revealed resistance to different classes of antibiotics in strain SOA01. Conclusion: Brucella anthropi SOA01 is a multidrug-resistant strain. Several AMR and VF genes were identified in the genome, revealing the potential threat posed by this pathogen. The genome data generated in this study are likely to be useful in better understanding its AMR mechanisms, pathogenic potential and successful adaptation from its primary habitat of soil to the human system. Since it is often misidentified as Brucella melitensis or Brucella suis, genome characterisation and detailed understanding of its biology are crucial. © 2021 The Author(s)

Background & Aims: Bacterial infections can trigger the development of organ failure(s) and acute-on-chronic liver failure (ACLF). Geographic variations in bacteriology and clinical practice could lead to worldwide differences in ACLF epidemiology, phenotypes and associated outcomes. Herein, we aimed to evaluate regional differences in bacterial infection-related ACLF in patients with cirrhosis admitted to hospital. Methods: This post hoc analysis included 1,175 patients with decompensated cirrhosis (with bacterial infection on admission or nosocomial infection) from 6 geographic regions worldwide. Clinical, laboratory and microbiological data were collected from the diagnosis of infection. Patients were followed-up for organ failure(s) and ACLF development according to the EASL-CLIF criteria from enrolment to discharge/death. Results: A total of 333 patients (28%) had ACLF at diagnosis of infection, while 230 patients developed ACLF after diagnosis of infection, resulting in an overall rate of bacterial infection related-ACLF of 48%, with rates differing amongst different geographic regions (38% in Southern Europe vs. 75% in the Indian subcontinent). Bacterial infection related-ACLF more frequently developed in younger patients (55 ± 13 vs. 58 ± 14 years), males (73% vs. 62%), patients with alcohol-related cirrhosis (59% vs. 45%) and those with a higher baseline MELD score (25 ± 11 vs. 16 ± 5) (all p

Tuberculosis (TB) continues to be the leading cause of deaths due to its persistent drug resistance and the consequent ineffectiveness of anti-TB treatment. Recent years witnessed huge amount of sequencing data, revealing mutations responsible for drug resistance. However, the lack of an up-to-date repository remains a barrier towards utilization of these data and identifying major mutations-associated with resistance. Amongst all mutations, non-synonymous mutations alter the amino acid sequence of a protein and have a much greater effect on pathogenicity. Hence, this type of gene mutation is of prime interest of the present study. The purpose of this study is to develop an updated database comprising almost all reported substitutions within the Mycobacterium tuberculosis (M.tb) drug target genes rpoB, inhA, katG, pncA, gyrA and gyrB. Various bioinformatics prediction tools were used to assess the structural and biophysical impacts of the resistance causing non-synonymous single nucleotide polymorphisms (nsSNPs) at the molecular level. This was followed by evaluating the impact of these mutations on binding affinity of the drugs to target proteins. We have developed a comprehensive online resource named MycoTRAP-DB (Mycobacterium tuberculosis Resistance Associated Polymorphisms Database) that connects mutations in genes with their structural, functional and pathogenic implications on protein. This database is accessible at http://139.59.12.92. This integrated platform would enable comprehensive analysis and prioritization of SNPs for the development of improved diagnostics and antimycobacterial medications. Moreover, our study puts forward secondary mutations that can be important for prognostic assessments of drug-resistance mechanism and actionable anti-TB drugs.

Tyrosine kinase inhibitors (TKIs) are important in managing lymphoid malignancies by targeting B-cell receptor signaling pathways. Entospletinib (GS-9973) is an oral, selective inhibitor of spleen tyrosine kinase (Syk), currently in the phase II clinical trials for the treatment of chronic lymphocytic leukemia. Syk is abundantly present in the cells of hematopoietic lineage that mediates cell proliferation, differentiation, and adhesion. In this current study, we evaluated the efficacy of GS-9973 to overcome multidrug resistance (MDR) due to the overexpression of the ABCG2 transporter in the non-small cell lung cancer (NSCLC) cell line, NCI-H460/MX20. In vitro, 3 μM of GS-9973 reversed the drug resistance of NCI-H460/MX20 cell line to mitoxantrone or doxorubicin. GS-9973, at 3 μM reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the protein level but did not alter the ABCG2 mRNA expression and subcellular localization of the ABCG2 protein compared to drug-resistant cells incubated with the vehicle. GS-9973 produced a moderate concentration-dependent increase in the ATPase activity of ABCG2 (EC50 = 0.42 µM) and molecular docking data indicated that GS-9973 had a high affinity (-10.226 kcal/mol) for the substrate-binding site of ABCG2. Finally, HPLC analysis proved that the intracellular concentration of GS-9973 is not significantly different in both parental and resistant cell lines. In conclusion, our study suggests that in vitro, GS-9973 in combination with certain anticancer drugs, represent a strategy to overcome ABCG2-mediated MDR cancers.

Although many drugs that targeted the specific features of leukemia stem cells (LSCs) have substantial application in the clinical treatment of leukemia, the LSCs relapsed and caused drug-resistant leukemia. Therefore, it is necessary to identify the unique features of LSCs in relapsing and drug-resistant leukemia and also to explore the drugs that directed at these features. Our clinical data have indicated that relapsed patients with acute myeloid leukemia have more abundant proportion of LSCs with enhanced breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) expression when compared to the untreated patients. The results showed that compared with LSCs derived from sensitive K562 cells, LSCs from drug-resistant K562/ADM cells have much higher chemotherapeutic resistance, and so we termed these cells as “drug-resistant LSCs”. Subsequently, aberrant activation of NF-κB pathway in drug-resistant LSCs was further using gene chip analysis. Also, parthenolide (PTL), which is a specific NF-κB inhibitor, effectively eliminated drug-resistant LSCs and enhanced the sensitivity of K562/ADM cells to doxorubicin-induced apoptosis by down-regulating NF-κB pathway-mediated P-gp expression. These findings make the research area of LSCs more abundant and provide a potential therapeutic strategy for the treatment of refractory and relapsed leukemia.

Objectives: The aim of this study was to examine how the concentrated delivery of less effective antibiotics, such as the β-lactam penicillin G, by linkage to nanoparticles (NPs), could influence the killing efficiency against various pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and other multidrug resistant (MDR) strains. Methods: The β-lactam antibiotic penicillin G (PenG) was passively sorbed to fluorescent polystyrene NPs (20 nm) that were surface-functionalized with carboxylic acid (COO−-NPs) or sulfate groups (SO4−-NPs) to form a PenG-NP complex. Antimicrobial activities of PenG-NPs were evaluated against Gram-negative and Gram-positive bacteria, including antibiotic resistant strains. Disc diffusion, microdilution assays and live/dead staining were performed for antibacterial assessments. Results: The results showed that bactericidal activities of PenG-NP complexes were statistically significantly (P < 0.05) enhanced against Gram-negative and Gram-positive strains, including MRSA and MDR strains. Fluorescence imaging verified that NPs comigrated with antibiotics throughout clear zones of MIC agar plate assays. The increased bactericidal abilities of NP-linked antibiotics are hypothesized to result from the greatly increased densities of antibiotic delivered by each NP to a given bacterial cell (compared with solution concentrations of antibiotic), which overwhelms the bacterial resistance mechanism(s). Conclusions: As a whole, PenG-NP complexation demonstrated a remarkable activity against different pathogenic bacteria, including MRSA and MDR strains. We term this the ‘grenade hypothesis’. Further testing and development of this approach will provide validation of its potential usefulness for controlling antibiotic-resistant bacterial infections.

Objectives To study the clinical profile, complications, antibiotic resistance pattern, treatment, and outcome of out-born neonates with Acinetobacter spp. sepsis admitted in Pediatric emergency of a tertiary care hospital in North India. Methods In this subgroup analysis of a prospective study (conducted over 1 y, February 2018 through January 2019), neonates with Acinetobacter spp. sepsis were included. The data collection included demographic details, clinical features, pre-referral treatment, complications, antibiotic resistance pattern, treatment, and final outcome. Results Acinetobacter spp. accounted for 10.6% (43/406) of all isolates and 22.7% (43/189) of Gram-negative isolates. The median (IQR) age at presentation was 1 (1–2) d, 2/3rd were male, and 46.5% were preterm. All were admitted in peripheral hospitals before referral to authors’ centre and all received intravenous antibiotics and fluids. The resistance to different antibiotics was: Ciprofloxacin 82%, cephalosporins 78–100%, amikacin 75%, pipercillin-tazobactum 62%, carbapenems 50–85%, chloramphenicol 83%, and tetracycline 50–60%. All isolates were sensitive to colistin. The survival rate was 37.2% (n = 16) and 62.8% (n = 27) had poor outcome [death and Left against medical advice (LAMA)]. Higher proportion of neonates with Acinetobacter sepsis had septic shock, multi-organ dysfunctional syndrome (MODS), and disseminated intravascular coagulation (DIC); and higher proportion required mechanical ventilation, vasoactive drugs, and had poor outcome compared to those with sepsis due to other organisms. Conclusions Acinetobacter spp. accounts for high burden of sepsis among out-born neonates and is associated with alarmingly high resistance to cephalosporins, fluroquinolones, aminoglycosides, pipercillin-tazobactum, tetracyclines, and carbapenems. Neonates with Acinetobacter spp. sepsis had higher rates of complications, requirement of mechanical ventilation and vasoactive drugs, and poor survival.

Killing or inhibiting the growth of pathogenic microorganisms in vivo has been made possible by the revolutionary discovery of antimicrobial substances that are tolerated by the human body [1,2]. I...

Escherichia coli sequence type (ST) 131 is of concern because it can acquire antimicrobial resistance and cause extraintestinal infections. E. coli ST131-H22 sublineage appears capable of being transmitted to humans through poultry. We report on multidrug-resistant ST131-H22 poultry isolates in Brazil closely related to international human and poultry isolates.

The objective: to study the structure of Beijing genotype family of Mycobacterium tuberculosis in Omsk Region, Western Siberia. Subjects and methods : 325 strains of M. tuberculosis were studied, all of them were isolated from tuberculosis patients in Omsk Region. Strains were tested to identify if they belonged to Beijing genotype, B 0/W 148 and 9 4-3 2. Locus NTF and region RD 181 were assessed. Ancient Beijing strains were analyzed by spoligotyping and MIR U - V NT R -t yped at 12 loci, and the obtained profiles were compared with the international database of SITVIT _ WEB . Results . Among 204 (62.7%) strains of M. tuberculosis from Beijing family, 176 (86.3%) of strains belonged to the modern sublineage of 9 4-3 2 (60.7%) and B 0/W 148 (25.0%) clusters. Inside 9 4-3 2 cluster, it was found that 13 (10.5%) strains belonged to CAO group. Strains of the ancient Beijing sublineages of RD 181[+] and RD 181[-] were detected in 3.4% ( 7/2 04) and 10.3% (2 1/2 04) cases, respectively. All of Beijing RD 181[+] strains had SIT 269 spoligoprofile. The majority of strains of the ancient sublineage were represented by the MIRU types – MIT 135 and MIT 642. Conclusion. In Omsk Region of Western Siberia, strains of the modern sublineage prevail in the population of M. tuberculosis of Beijing family, the prevalence of ancient strains of this family is (13.7%). The high level of multiple drug resistance is typical of strains of the modern sublineage of B 0/W 148, 9 4-3 2 CAO clusters and strains of the ancient Beijing sublineage.

Objectives: Objective: The aim of this study is to describe the epidemiological surveillance of Carbapenem-Resistant Enterobacteriaceae (CRE) effected at the Neurological Hospital Carlo Besta, Milano, Italy. The primary causes in our division of multidrug-resistant (MDRs) infections in 2016 were K. pneumoniae and A. baumannii; the preventive actions have ensured the reduction of their incidence in 2017 and allowed us to discover the microbiological sentinel events (SE) and start appropriate precautionary behavior and isolation. Methods: The setting is inpatient care with MDR infections and the aim of the procedure is to formalize the management mode, reporting and transmission of SE, collected at the end of study period for previous years from January 1st, 2015 to December 31st, 2017. Results: It should be noted that of a total of 5772 hospitalizations detected in 2016 there was an increase in rate of A. baumannii compared to the rate found in 2015 for 5679 hospitalizations and in 2017 for 5682 hospitalizations; the same trend was observed for K. pneumoniae. For as regards P. aeruginosa carbapenemase producing or MDR, the rates were lower in 2016 and in 2017. Conclusions: The main goals that we achieved in the three years considered are the decreased incidence of healthcare-associated infections (HAIs) and the decreased incidence of multidrug-resistant (MDR) microorganisms. These actions were of course associated with the control of antibiotic pressure and a careful stewardship. The strict surveillance of HAIs is fundamental to control the growing reservoir within hospital communities. J Microbiol Infect Dis 2020; 10(1):1-9.

Purpose: A retrospective study was first performed to assess the multidrug resistant (MDR) pathogen in severe acute pancreatitis (SAP) patients who were treated using the step-up approach. We aim to assess the risk factors between MDR pathogen and potential covariates in SAP patients. Methods: The clinical data of 51 SAP patients who were treated from June, 2013 to December, 2016 were retrospectively collected. A total of 23 patients in the MDR group and 28 patients in the non-MDR group were reviewed. The risk factors for MDR pathogen-induced infections in SAP patients were analyzed. Results: Hyperlipidemia was the leading cause of SAP in our study. The mean duration of hospital stay was significantly longer in the patients with MDR pathogen infections (P=0.0135). The hospitalization expenses of MDR group were much higher than those in non-MDR group. The mortality of MDR group (56.5%) was higher than that in non-MDR group (28.6%) (P=0.0436). Gram-negative isolates (63.8%) were commonly detected in SAP patients. Acinetobacter baumannii was the most common MDR pathogens. Systemic disease (P = 0.0136), initial use of carbapenem (P = 0.0438), and open necrosectomy (P = 0.0002) were the potential risk factors for MDR pathogen-induced infections in SAP. Furthermore, the logistic regression analysis revealed that open necrosectomy was the independent variable for MDR infections (OR: 15.6, 95% CI: 2.951–82.469, P = 0.0012). Conclusions: MDR pathogen-induced infections were common in SAP patients and Acinetobacter baumannii was the main pathogen. Meanwhile, open necrosectomy was the independent risk factor for the infection of MDR pathogen.

Chemotherapy resistance represents a major obstacle for the treatment of patients with breast cancer (BC) and greatly restricts the therapeutic effect of the first-line chemotherapeutic agent doxorubicin (DOX). The present study aimed to investigate the feasibility of the recombinant dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor in reversing the DOX resistance of BC. Both DOX-resistant human breast carcinoma cell lines exhibited a multidrug resistance (MDR) phenotype. The ability of the dual-target MDM2/MDMX inhibitor in reversing doxorubicin resistance was subsequently verified, (9.15 and 13.92 - fold reversal indexes) respectively. We observed that the MDM2/MDMX inhibitor in combination with DOX could suppress proliferation, promote cell cycle arrest and induce apoptosis. In addition, it was capable of reducing rhodamine123 efflux in DOX-resistance BC cell lines and further played a key role in BC nude mice model. The groups that were treated with the combination of the drugs had decreased P-glycoprotein/multidrug resistance-associated protein/cdc 2/Bcl-2 expression and increased CyclinB1/Bax expression. These effects were caused due to activation of the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)/TAK1/p38 mitogen-activated protein kinase (MAPK) signaling pathway, as shown by small interfering RNA (siRNA) silencing and immumohistochemical staining of BC tissue sections. Furthermore, high MDM2/MDMX expression was positively associated with weak TAB1 expression in BC patients. Therefore, the recombinant dual-target MDM2/MDMX inhibitor could reverse doxorubicin resistance via the activation of the TAB1/TAK1/p38 MAPK pathway in wild-type p53 multidrug-resistant BC.

Tuberculosis disease (TB), caused by Mycobacterium tuberculosis, is a major public health issue in Thailand. The high prevalence of modern Beijing (Lineage 2.2.1) strains has been associated with multi- and extensively drug-resistant infections (MDR-, XDR-TB), complicating disease control. The impact of rarer proto-Beijing (L2.1) strains is less clear. In our study of thirty-seven L2.1 clinical isolates spanning thirteen years, we found a high prevalence of XDR-TB cases (32.4%). With ≤ 12 pairwise SNP distances, 43.2% of L2.1 patients belong to MDR-TB or XDR-TB transmission clusters suggesting a high level of clonal expansion across four Thai provinces. All XDR-TB (100%) were likely due to transmission rather than inadequate treatment. We found a 47 mutation signature and a partial deletion of the fadD14 gene in the circulating XDR-TB cluster, which can be used for surveillance of this rare and resilient M. tuberculosis strain-type that is causing increasing health burden. We also detected three novel deletion positions, a deletion of 1285 bp within desA3 (Rv3230c), large deletions in the plcB, plcA, and ppe38 gene which may play a role in the virulence, pathogenesis or evolution of the L2.1 strain-type.

Klebsiella pneumoniae is an opportunistic pathogen that is very difficult to treat mainly due to its high propensity to acquire complex resistance traits. Notably, multidrug resistance (MDR)-Klebsiella pneumoniae (KP) infections are responsible for 22%–72% of mortality among hospitalized and immunocompromised patients. Although treatments with new drugs or with combined antibiotic therapies have some degree of success, there is still the urgency to investigate and develop an efficient approach against MDR-KP infections. In this study, we have evaluated, in an in vitro model of human macrophages, the efficacy of a combined treatment consisting of apoptotic body-like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) and φBO1E, a lytic phage specific for the major high-risk clone of KPC-positive MDR-KP. Results show that ABL/PI5P did not affect in a direct manner KKBO-1 viability, being able to reduce only the intracellular KKBO-1 bacterial load. As expected, φBO1E was effective mainly on reducing extracellular bacilli. Importantly, the combination of both treatments resulted in a simultaneous reduction of both intracellular and extracellular bacilli. Moreover, the combined treatment of KKBO-1-infected cells reduced proinflammatory TNF-α and IL-1β cytokines and increased anti-inflammatory TGF-β cytokine production. Overall, our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with MDR pathogens such as MDR-KP.

BackgroundOur interconnected world and the ability of bacteria to quickly swap antibiotic resistance genes (ARGs) make it particularly important to establish the epidemiological links of multidrug resistance (MDR) transfer between wastewater treatment plant (WWTP)- and human/animal-associated bacteria, under the One Health framework. However, evidence of ARGs exchange and potential factors that contribute to this transfer remain limited.ResultsHere, by combining culture-based population genomics and genetic comparisons with publicly available datasets, we reconstructed the complete genomes of 82 multidrug-resistant isolates from WWTPs and found that most WWTP-associated isolates were genetically distinct from their closest human/animal-associated relatives currently available in the public database. Even in the minority of lineages that were closely related, WWTP-associated isolates were characterized by quite different plasmid compositions. We identified a high diversity of circular plasmids (264 in total, of which 141 were potentially novel), which served as the main source of resistance, and showed potential horizontal transfer of ARG-bearing plasmids between WWTP- and humans/animal-associated bacteria. Notably, the potentially transferred ARGs and virulence factors (VFs) with different genetic backgrounds were closely associated with flanking insertion sequences (ISs), suggesting the importance of synergy between plasmids and ISs in mediating a multilayered hierarchical transfer of MDR and potentiating the emergence of MDR-hypervirulent clones.ConclusionOur findings advance the current efforts to establish potential epidemiological links of MDR transmission between WWTP- and human/animal-associated bacteria. Plasmids play an important role in mediating the transfer of ARGs and the IS-associated ARGs that are carried by conjugative plasmids should be prioritized to tackle the spread of resistance.

The present White Paper has been developed in the course of the Made4You/Careables.org H2020 project. Its purpose is to map common legal challenges of Open-Source Hardware for healthcare. It addresses privacy and data protection, intellectual property rights, liability, and medical devices legislation. The White Paper is addressed to makers, designers, and healthcare professionals involved in co-design as co-creation of open-source hardware healthcare solutions, fablabs, as well as DPOs and local networks for industry representatives. ispartof: Made4You - White Paper on Legal Guidelines status: Published online

YS and MT was supported by Proyectos de Investigación en Salud, Instituto de Salud Carlos III (PI19/01009) and (PI19/00878), respectively, cofinanced by the European Development Regional Fund (A way to achieve Europe, Operative Program Intelligent Growth 2014–2020).

Infectious disease is one of the greatest causes of morbidity and mortality worldwide, and with the emergence of antimicrobial resistance, the situation is worsening. In order to prevent this crisis, antimicrobial resistance needs to be monitored carefully to control the spread of multidrug-resistant bacteria. Therefore, in this study, we aimed to determine the prevalence of infection caused by Klebsiella pneumoniae and investigate the antimicrobial profile pattern of K. pneumoniae in the last eleven years. This retrospective study was conducted in a tertiary hospital in Makkah, Saudi Arabia. Data were collected from January 2011 to December 2021. From 2011 to 2021, a total of 61,027 bacterial isolates were collected from clinical samples, among which 14.7% (n = 9014) were K. pneumoniae. The antibiotic susceptibility pattern of K. pneumoniae revealed a significant increase in the resistance rate in most tested antibiotics during the study period. A marked jump in the resistance rate was seen in amoxicillin/clavulanate and piperacillin/tazobactam, from 33.6% and 13.6% in 2011 to 71.4% and 84.9% in 2021, respectively. Ceftazidime, cefotaxime, and cefepime resistance rates increased from 29.9%, 26.2%, and 53.9%, respectively, in 2011 to become 84.9%, 85.1%, and 85.8% in 2021. Moreover, a significant increase in the resistance rate was seen in both imipenem and amikacin, with an average resistance rate rise from 6.6% for imipenem and 11.9% for amikacin in 2011 to 59.9% and 62.2% in 2021, respectively. The present study showed that the prevalence and drug resistance of K. pneumoniae increased over the study period. Thus, preventing hospital-acquired infection and the reasonable use of antibiotics must be implemented to control and reduce antimicrobial resistance.

The widespread and indiscriminate use of broad-spectrum antibiotics leads to microbial resistance, which causes major problems in the treatment of infectious diseases. However, advances in nanotechnology have opened up new domains for the synthesis and use of nanoparticles against multidrug-resistant pathogens. The traditional approaches for nanoparticle synthesis are not only expensive, laborious, and hazardous but also have various limitations. Therefore, new biological approaches are being designed to synthesize economical and environmentally friendly nanoparticles with enhanced antimicrobial activity. The current study focuses on the isolation, identification, and screening of metallotolerant fungal strains for the production of silver nanoparticles, using antimicrobial activity analysis and the characterization of biologically synthesized silver nanoparticles by X-ray diffraction (XRD) spectroscopy, energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM). In total, 11 fungal isolates were isolated and screened for the synthesis of AgNPs, while the Penicillium notatum (K1) strain was found to be the most potent, demonstrating biosynthetic ability. The biologically synthesized silver nanoparticles showed excellent antibacterial activity against the bacteria Escherichia coli (ATCC10536), Bacillus subtilis, Staphylococcus aureus (ATCC9144), Pseudomonas aeruginosa (ATCC10145), Enterococcus faecalis, and Listeria innocua (ATCC13932). Furthermore, three major diffraction peaks in the XRD characterization, located at the 2θ values of 28.4, 34.8, 38.2, 44, 64, and 77°, confirmed the presence of AgNPs, while elemental composition analysis via EDX and spherical surface topology with a scanning electron microscope indicated that its pure crystalline nature was entirely composed of silver. Thus, the current study indicates the enhanced antibacterial capability of mycologically synthesized AgNPs, which could be used to counter multidrug-resistant pathogens.

Malaysia Klang River covering states of Selangor and Kuala Lumpur, has been severely polluted from urbanization, industrialization and increased hospitals. By serving as reservoir for bacterial gene transformation, Multidrug Resistant (MDR) strains especially Extended Spectrum Beta Lactamase (ESBL) resistant strains of Klebsiella pneumoniae (K. pneumoniae) have increased. To identify MDR K.pneumoniae; phenotypically determine ESBL resistant traits and molecular characterize these strains, 50 water samples were collected along Klang River and their physicochemical properties were determined. Gram-negative K. pneumoniae isolates were tested for multidrug and ESBL resistance using Kirby-Bauer method. DNA of ESBL resistant K. pneumoniae were extracted and amplified using Polymerase Chain Reaction (PCR); and conferred using blaTEM and blaCTX-M genes. Through β-lactamase gene sequencing and docking studies, the effectiveness of drugs against ESBL resistant K.pneumoniae were determined. A total of 31 (62.0%) K.pneumoniae were isolated with 12 (38.7%) positive MDR strains and 5 (41.7%) ESBL resistant strains. Only 1 (20%) blaTEM gene and 4 (80%) blaCTX-M genes were detected with Ceftriaxone (CRO) as the most effective drug showing highest binding energy (-9.2Kcal/mol) against ESBL resistant K. pneumoniae. With high prevalence of CTX-M-type ESBL resistant K.pneumoniae in Klang River, effective environmental and antibiotic controls should be adopted.

Background Bloodstream infections (BSIs) caused by multidrug-resistant (MDR)-Pseudomonas aeruginosa are associated with poor clinical outcomes, at least partly due to delayed appropriate antimicrobial therapy. The characteristics of MDR-P. aeruginosa bloodstream isolates have not been evaluated in Qatar. Our study aimed to examine in vitro susceptibility, clinical and molecular characteristics, and mechanisms of resistance of MDR-P. aeruginosa bloodstream isolates from Qatar. Materials and methods We included all MDR-P. aeruginosa isolated from blood cultures taken between October 2014 and September 2017. Blood cultures were processed using BD BACTEC™ FX automated system. BD Phoenix™ was used for identification, Liofilchem® MIC Test Strips for MIC determination. Whole-genome sequencing was performed using the Illumina-HiSeq-2000. Results Out of 362 P. aeruginosa bloodstream isolates, 16 (4.4%) were MDR. The median patient age was 55 years (range 43–81) and all patients presented with septic shock. Most patients received meropenem (12/16) and/or colistin (10/16). Clinical response was achieved in eight patients, and five patients died within 30-days. MDR-P. aeruginosa isolates belonged to 13 different sequence types. All isolates were non-susceptible to cefepime and ciprofloxacin. The most active agents were colistin (16/16) and aztreonam (10/16). Seven isolates produced blaVIM, and four possessed genes encoding extended-spectrum β-lactamases. Aminoglycoside modifying enzymes were present in 15/16, transferable qnr-mediated quinolone resistance gene was detected in 3/16, and the novel ciprofloxacin modifying enzyme CrpP-encoding gene in one isolate. Conclusion MDR-P. aeruginosa BSIs are relatively uncommon in Qatar but are highly resistant, harbour multiple resistance genes, and are commonly associated with unfavourable clinical outcomes. Colistin was the only agent with consistent activity against the study isolates.Key messagesMDR-P. aeruginosa constituted

The efforts made in the last decade regarding the molecular landscape of acute myeloid leukemia (AML) have created the possibility of obtaining patients’ personalized treatment. Indeed, the improvement of accurate diagnosis and precise assessment of minimal residual disease (MRD) increased the number of new markers suitable for novel and targeted therapies. This progress was obtained thanks to the development of molecular techniques starting with real-time quantitative PCR (Rt-qPCR) passing through digital droplet PCR (ddPCR) and next-generation sequencing (NGS) up to the new attractive metabolomic approach. The objective of this surge in technological advances is a better delineation of AML clonal heterogeneity, monitoring patients without disease-specific mutation and designing customized post-remission strategies based on MRD assessment. In this context, metabolomics, which pertains to overall small molecules profiling, emerged as relevant access for risk stratification and targeted therapies improvement. In this review, we performed a detailed overview of the most popular modern methods used in hematological laboratories, pointing out their vital importance for MRD monitoring in order to improve overall survival, early detection of possible relapses and treatment efficacy.

Background. Stenotrophomonas maltophilia is an emerging pathogen classified as a public health concern, that infects critically ill patients and has expressed resistance against antimicrobial therapy. The aim of this study was to examine the epidemiological pattern, resistance characteristics and clinical outcomes of S. maltophilia infections in hospitalized patients. Methods. The study included 393 S. maltophilia isolates from different clinical specimens as well as the clinical data of 209 Intensive Care Unit (ICU) patients. The patients’ data were obtained from medical and laboratory files. Descriptive statistics and a univariate analysis were used to report and compare the demographics, clinical data, and outcomes. Results. The S. maltophilia was mostly isolated from the respiratory specimens of ICU patients. The adult patients were more likely to develop serious infections and worse outcomes than were pediatric patients. The most common co-infecting pathogens were SARS-CoV2 and Pseudomonas aeruginosa. The death rate was 44.5% and increased to 47.1% in the case of a respiratory infection. Septic shock was the most significant predictor of mortality. Older age and mechanical ventilation were independent and significant risk factors that worsened the outcomes in patients with respiratory infections. Conclusions. The identification of S. maltophilia as a threat highlights the importance of surveillance studies in this region.

Bacterial urinary tract infection (UTI) is a common diagnosis in companion animal practice and is one of the leading reasons for antimicrobial prescriptions. We analysed 1862 samples from the urinary tract of dogs and cats, submitted to a veterinary microbiological diagnostic laboratory in 2019 and 2020 in Germany. Susceptibility of 962 uropathogenic isolates to 15 antimicrobials, suggested as first- and second-line treatment options for UTI, was determined according to CLSI recommendations. Bacterial growth of uropathogens was detected in 43.9% of dog and in 38.5% of cat samples. Escherichia (E.) coli was the most frequently isolated pathogen (48.4%), followed by Enterococcus spp. (11.9%) and coagulase-positive staphylococci (CoPS; 11.5%). Females were more likely to exhibit a positive microbiological culture. Regarding first-line antibiotics, 93.4% of the most commonly isolated uropathogenic species were susceptible to the first-line antibiotics amoxicillin/clavulanic acid (AMC) and 87.6% to trimethoprim-sulfamethoxazole (SXT), while 76.1% showed decreased susceptibility to ampicillin (AMP). Multidrug resistance (MDR) was detected in 11.9% of E. coli, 50.4% of enterococci, and 42.7% of CoPS; 90.6% of these isolates were susceptible to nitrofurantoin (NIT). Our data indicate that empiric treatment of UTI with AMC or SXT could be recommended and is preferable to treatment with AMX. NIT should be considered for the treatment of MDR uropathogens.

Klebsiella pneumoniae is an increasingly important hospital pathogen. Classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) are two distinct evolutionary genetic lines. The recently ongoing evolution of K. pneumoniae resulted in the generation of hybrid hvKP-MDR strains. K. pneumoniae distinct isolates (n = 70) belonged to 20 sequence types with the prevalence of ST395 (27.1%), ST23 (18.6%), ST147 (15.7%), and ST86 (7.1%), and 17 capsular types with the predominance of K2 (31.4%), K57 (18.6%), K64 (10.0%), K1 (5.7%) were isolated from patients of the Moscow neurosurgery ICU in 2014–2019. The rate of multi-drug resistant (MDR) and carbapenem-resistant phenotypes were 84.3% and 45.7%, respectively. Whole-genome sequencing of five selected strains belonging to cKp (ST395K47 and ST147K64), hvKp (ST86K2), and hvKp-MDR (ST23K1 and ST23K57) revealed blaSHV, blaTEM, blaCTX, blaOXA-48, and blaNDM beta-lactamase genes; acr, oqx, kpn, kde, and kex efflux genes; and K. pneumoniae virulence genes. Selective pressure of 100 mg/L ampicillin or 10 mg/L ceftriaxone induced changes of expression levels for named genes in the strains belonging to cKp, hvKp, and hybrid hvKp-MDR. Obtained results seem to be important for epidemiologists and clinicians for enhancing knowledge about hospital pathogens.

Multidrug resistance (MDR) is a major concern in battling infectious bacterial diseases. The overuse of antibiotics contributes to the emergence of resistance by eradicating the drug-sensitive strains, leaving behind the resistant strains that multiply without any competition. Nanoparticles are becoming popular as novel antimicrobial agents that follow a different mode of action from standard antibiotics and are therefore desirable against MDR bacteria. In this study, we synthesized carbon dots from different precursors including glucosamine HCL (GlcNH2·HCl) and 4,7,10-trioxa-1,13-tridecanediamine (TTDDA, and studied their antimicrobial effects in a diverse list of Gram-negative bacteria including Escherichia coli, Pseudomonas syringae, Salmonella enterica subsp. enterica serovar Typhimurium, Pectobacterium carotovorum, Agrobacterium tumefaciens, and Agrobacterium rhizogenes. We demonstrated the antimicrobial properties of these carbon dots against these bacteria and provided the optimum concentration and incubation times for each bacterial species. Our findings indicated that not all carbon dots carry antimicrobial properties, and there is also a variation between different bacterial species in their resistance against these carbon dots.

This research aimed to investigate the presence and transferability of the extended-spectrum β-lactamase resistance genes to identify the genetic context of multi-drug resistant (MDR) loci in two Escherichia coli plasmids from livestock and poultry breeding environment. MICs were determined by broth microdilution. A total of 137 E. coli resistant to extended-spectrum β-lactam antibiotics were screened for the presence of the ESBL genes by PCR. Only two E. coli out of 206 strains produced carbapenemases, including strain 11011 that produced enzyme A, and strain 417957 that produced enzyme B. The genes were blaKPC and blaNDM, respectively. The plasmids containing blaCTX–M were conjugatable, and the plasmids containing carbapenem resistance gene were not conjugatable. Six extended-spectrum β-lactamase resistance genes were detected in this research, including blaTEM, blaCTX–M, blaSHV, blaOAX–1, blaKPC, and blaNDM, and the detection rates were 94.89% (130/137), 92.7% (127/137), 24.81% (34/137), 20.43% (28/137), 0.72% (1/137), and 0.72% (1/137), respectively. Two conjugative lncFII multi-resistance plasmids carrying blaCTX–M, p11011-fosA and p417957-CTXM, were sequenced and analyzed. Both conjugative plasmids were larger than 100 kb and contained three accessory modules, including MDR region. The MDR region of the two plasmids contained many antibiotic resistance genes, including blaCTX–M, mph (A), dfrA17, aadA5, sul1, etc. After transfer, both the transconjugants displayed elevated MICs of the respective antimicrobial agents. A large number of resistance genes clusters in specific regions may contribute to the MDR profile of the strains. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterobacteriaceae through inter-replicon gene transfer. Our study provides beta-lactam resistance profile of bacteria, reveals the prevalence of β-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province, and enriches the research on IncFII plasmids containing blaCTX–M.

Tuberculosis causes more than 1.2 million deaths each year. Worldwide, it is the first cause of death by a single infectious agent. The emergence of drug-resistant strains has limited pharmacological treatment of the disease and today, new drugs are urgently needed. Semi-synthetic mulinanes have previously shown important activity against multidrug-resistant (MDR) Mycobacterium tuberculosis. In this investigation, a new set of semi-synthetic mulinanes were synthetized, characterized, and evaluated for their in vitro activity against three drug-resistant clinical isolates of M. tuberculosis: MDR, pre-extensively Drug-Resistant (pre-XDR), and extensively Drug-Resistant (XDR), and against the drug-susceptible laboratory reference strain H37Rv. Derivative 1a showed the best anti-TB activity (minimum inhibitory concentration [MIC] = 5.4 µM) against the susceptible strain and was twice as potent (MIC = 2.7 µM) on the MDR, pre-XDR, and XDR strains and also possessed a bactericidal effect. Derivative 1a was also tested for its anti-TB activity in mice infected with the MDR strain. In this case, 1a produced a significant reduction of pulmonary bacilli loads, six times lower than the control, when tested at 0.2536 mg/Kg. In addition, 1a demonstrated an adjuvant effect by shortening second-line chemotherapy. Finally, the selectivity index of >15.64 shown by 1a when tested on Vero cells makes this derivative an important candidate for future studies in the development of novel antitubercular agents.

A total of 394 strains of staphylococci found in humans and pets in Novosibirsk, Siberian Russia, were characterized in terms of antibiotic resistance and corresponding genes. Two coagulase-positive and 17 coagulase-negative species were identified. The majority of isolates, with the exception of S. haemolyticus and hospital S. epidermidis isolates, were sensitive to most of the tested antibiotics, and isolates from pets displayed the lowest level of resistance. Nevertheless, methicillin-resistant (MRS) and/or multidrug-resistant (MDR) isolates were found in all prevailed species, including coagulase-negative. A set of genes corresponding to the detected resistance was identified: mecA (beta-lactam resistance), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, ant(4′)-Ia (aminoglycoside-modifying enzymes), ermA/ermC, and msrA (macrolide resistance). Complete genome analysis for ten MDR S. epidermidis and five MDR S. haemolyticus isolates revealed additional antibiotic resistance genes mphC, qacA/qacB, norA, dfrC/dfrG, lnuA, BseSR, and fosB. NorA, dfrC, and fosB were present in all S. epidermidis genomes, whereas mphC and msrA were identified in all S. haemolyticus ones. All investigated MDR S. epidermidis and four of five S. haemolyticus strains were moderate or strong biofilm producers, whereas multiple genes responsible for this function and for virulence and pathogenicity were identified mostly in S. epidermidis, but were less frequently represented in S. haemolyticus.

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100–12.5 µM) and HK (100–12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

Background Gender differences among patients with drug resistant tuberculosis (DRTB) and HIV co-infection could affect treatment outcomes. We compared characteristics and treatment outcomes of DRTB/HIV co-infected men and women in Uganda. Methods We conducted a retrospective chart review of patients with DRTB from 16 treatment sites in Uganda. Eligible patients were aged ≥ 18 years, had confirmed DRTB, HIV co-infection and a treatment outcome registered between 2013 and 2019. We compared socio-demographic and clinical characteristics and tuberculosis treatment outcomes between men and women. Potential predictors of mortality were determined by cox proportional hazard regression analysis that controlled for gender. Statistical significance was set at p Results Of 666 DRTB/HIV co-infected patients, 401 (60.2%) were men. The median (IQR) age of men and women was 37.0 (13.0) and 34.0 (13.0) years respectively (p 3 increment). Conclusion Mortality was higher among men than women with DRTB/HIV co-infection which could be explained by several sociodemographic and clinical differences.