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247900.9673Down-regulatory effects of green coffee extract on las I and las R virulence-associated genes in Pseudomonas aeruginosa. BACKGROUND: Antibiotic resistant strains of Pseudomonas aeruginosa are the cause of Gram negative nosocomial infections especially among the immunosuppressed patients. The bacteria contains las I and las R genes that play very important roles in the pathogenesis and mechanisms of aggression. These genes can be influenced by the quorum sensing (QS) system and such mechanism is becoming clinically important worldwide. This study aimed to investigate the preventive effects of green coffee extract (GCE) on the expression of pathogenesis-related genes, las I and las R in P. aeruginosa. METHODS: A total of fifty four P. aeruginosa strains were isolated out of 100 clinical samples collected from the infectious wards in different hospitals (Tehran province) using conventional microscopic and biochemical methods. Susceptibility of the isolates to different antibiotics, GCE and chlorogenic acid were elucidated. Multiplex polymerase chain reaction (PCR) and real-time PCR were performed to detect and quantify the expression levels of las I and las R genes. The presence of chlorogenic acid in GCE was confirmed by HPLC. RESULTS: Antibiotic susceptibility tests revealed multidrug resistance among the clinical isolates of those 40 strains were resistant to ciprofloxacin (74.07%), 43 to ceftazidime (79.26%), 29 to amikacin (53.7%), 42 to ampicillin (77.77%), 17 to colistin (31.48%), 40 to gentamicin (74.77%), and 50 to piperacillin (92.59%). PCR outcomes exhibited that the frequency of las I and las R genes were 100% in resistant and sensitive strains isolated from clinical and standard strains of P. aeruginosa (ATCC 15449). Real-time PCR analyses revealed that GCE significantly prevented the expression of las I and las R genes in P. aeruginosa. GCE at concentration level as low as 2.5 mg/mL could prevent the expression of lasI and lasR genes in P. aeruginosa clinical isolates. CONCLUSION: The presence and expression levels of las I and las R genes in P. aeruginosa isolates were investigated when the bacteria was exposed to GCE. Our results tend to suggest that genes involved in pathogenesis of:Pseudomonas aeruginosa are down regulated by quorum sensing effect of chlorogenic acid and therefore GCE could be useful as an adjuvant in combating multidrug resistance strains of Pseudomonas aeruginosa.201931187452
209710.9672Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae. BACKGROUND: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. METHOD: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin-tazobactam (Pip-Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm(2) for 30 min aPDI. RESULTS: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. CONCLUSIONS: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.202133922077
140520.9657The threat of carbapenem resistance in Eastern Europe in patients with decompensated cirrhosis admitted to intensive care unit. BACKGROUND: Multidrug-resistant organisms are an increasing concern in patients with decompensated cirrhosis. AIM: We aimed to evaluate the prevalence of infections with carbapenem-resistant Enterobacteriaceae in patients with decompensated cirrhosis. METHODS: Patients with decompensated cirrhosis admitted to ICU were included. The isolated Enterobacteriaceae strains were tested for carbapenemase-producing genes using the Roche LightMix® Modular VIM/IMP/NDM/GES/KPC/OXA48-carbapenemase detection kit. RESULTS: 48 culture-positive infections were registered in 75 patients with acutely decompensated cirrhosis. Thirty patients contracted a second infection. 46% of bacteria isolated at admission and 60% of bacteria responsible for infections identified during ICU-stay were multiresistant. ESBL+ Enterobacteriaceae were predominant at admission, while carbapenem-resistance was dominant in both Enterobacteriaceae and Non-Fermenting-Gram-Negative Bacteria responsible for infections diagnosed during hospitalisation. OXA 48 or KPC type carbapenemases were present in 30% of the analyzed Enterobacteriaceae and in 40% of the phenotypically carbapenem-resistant Klebsiella pneumoniae strains. The length of ICU stay was a risk-factor for a second infection (p=0.04). Previous carbapenem usage was associated with occurence of infections with carbapenem-resistant Gram-negative bacteria during hospitalization (p=0.03). CONCLUSION: The prevalence of infections with carbapenem-resistant Enterobacteriaceae is high in patients with decompensated cirrhosis admitted to ICU. Carbapenemase-producing genes in Enterobacteriaceae in our center are bla(OXA-48) and bla(KPC).202235732546
140030.9656Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
219740.9649Antimicrobial susceptibility patterns of bacteria that commonly cause bacteremia at a tertiary hospital in Zambia. Background: Bloodstream infections and antimicrobial resistance cause global increases in morbidity and mortality. Aim: We evaluated the antimicrobial susceptibility patterns of bacteria that commonly cause bacteremia in humans. Materials & methods: We conducted a retrospective cross-sectional study at the University Teaching Hospitals in Lusaka, Zambia, using Laboratory Information Systems. Results: The commonest isolated bacteria associated with sepsis were Klebsiella pneumoniae. The distribution of bacteria associated with bacteremia in different wards and departments pneumonia. The distribution of bacteria associated with bacteremia in different wards and departments at University Teaching Hospitals was were statistically significant (χ2 = 1211.518; p < 0.001). Conclusion:K. pneumoniae, Escherichia coli, Pantoea agglomerans and Enterococcus species have developed high resistance levels against ampicillin, cefotaxime, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole and a very low resistance levels against imipenem and Amikacin.202033315486
636950.9647Association of furanone C-30 with biofilm formation & antibiotic resistance in Pseudomonas aeruginosa. BACKGROUND & OBJECTIVES: Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial bloodstream infections in humans. This study was aimed to explore the association of furanone C-30 with biofilm formation, quorum sensing (QS) system and antibiotic resistance in P. aeruginosa. METHODS: An in vitro model of P. aeruginosa bacterial biofilm was established using the standard P. aeruginosa strain (PAO-1). After treatment with 2.5 and 5 μg/ml of furanone C-30, the change of biofilm morphology of PAO-1 was observed, and the expression levels of QS-regulated virulence genes (lasB, rhlA and phzA2), QS receptor genes (lasR, rhlR and pqsR) as well as QS signal molecule synthase genes (lasI, rhlI, pqsE and pqsH) were determined. Besides, the AmpC expression was quantified in planktonic and mature biofilm induced by antibiotics. RESULTS: Furanone C-30 treatment significantly inhibited biofilm formation in a dose-dependent manner. With the increase of furanone C-30 concentration, the expression levels of lasB, rhlA, phzA2, pqsR, lasI, rhlI pqsE and pqsH significantly decreased in mature biofilm bacteria while the expression levels of lasR and rhlR markedly increased. The AmpC expression was significantly decreased in both planktonic and biofilm bacteria induced by imipenem and ceftazidime. INTERPRETATION & CONCLUSIONS: Furanone C-30 may inhibit biofilm formation and antibiotic resistance in P. aeruginosa through regulating QS genes. The inhibitory effect of furanone C-30 on las system appeared to be stronger than that on rhl system. Further studies need to be done with different strains of P. aeruginosa to confirm our findings.201829998876
219660.9646Antibiotic resistance profiles in Gram-negative bacteria causing bloodstream and urinary tract infections in paediatric and adult patients in Ndola District, Zambia, 2020-2021. BACKGROUND: Bloodstream infections (BSIs) and urinary tract infections (UTIs) caused by antibiotic resistant bacteria (ARB) have unfavourable treatment outcomes and negative economic impacts. OBJECTIVES: The main objective of this study was to determine antibiotic resistance profiles in Gram-negative bacteria (GNB) causing BSIs and UTIs. METHOD: A prospective study from October 2020 to January 2021 at Ndola Teaching Hospital and Arthur Davison Children's Hospital in the Ndola district, Zambia. Blood and urine samples collected from inpatients and outpatients presenting with fever and/or urinary tract infection symptoms were submitted for microbiological analysis. Pathogen identification and antibiotic susceptibility was determined by the automated VITEK 2 Compact machine. Resistance genes to commonly used antibiotics were determined using polymerase chain reaction. Data were analysed using SPSS version 28.0. RESULTS: One hundred and ten GNB were isolated, E. coli (45.5%) was predominant, with varying resistance profiles to different antibiotic classes. Resistance to third-generation cephalosporin was highest in Enterobacter cloacae (75%) and Klebsiella pneumoniae (71%), respectively. Emergence of carbapenem resistance was noted with the highest being 17% in Acinetobacter baumannii. Notably, the prevalence of multi-drug resistance was 63% and extensively drug-resistance was 32%. Resistance gene determinants identified included bla (CTX-M,) qnrA and bla (NDM). CONCLUSION: High level antibiotic resistance was observed in GNB known to be prevalent causative agents of BSIs and UTIs locally in Zambia. Improving microbiology diagnostic capacity, strengthening antimicrobial stewardship programs and enforcing infection prevention and control measures are of utmost importance in promoting rational use of antibiotics and preventing the spread and emergence of resistant pathogens.202540585877
140670.9644Multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers. BACKGROUND: Infected diabetic foot ulcer (IDFU) is a public health issue and the leading cause of non-traumatic limb amputation. Very few published data on IDFU exist in most West African countries. OBJECTIVE: The study investigated the aetiology and antibacterial drug resistance burden of IDFU in tertiary hospitals in Osun state, Nigeria, between July 2016 and April 2017. METHODS: Isolates were cultured from tissue biopsies or aspirates collected from patients with IDFU. Bacterial identification, antibiotic susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase and carbapenemase production were done by established protocols. Specific resistance genes were detected by polymerase chain reaction. RESULTS: There were 218 microorganisms isolated from 93 IDFUs, comprising 129 (59.2%) Gram-negative bacilli (GNB), 59 (27.1%) Gram-positive cocci and 29 (13.3%) anaerobic bacteria. The top five facultative anaerobic bacteria isolated were: Staphylococcus aureus (34; 15.6%), Escherichia coli (23; 10.6%), Pseudomonas aeruginosa (20; 9.2%), Klebsiella pneumoniae (19; 8.7%) and Citrobacter spp. (19; 8.7%). The most common anaerobes were Bacteroides spp. (7; 3.2%) and Peptostreptococcus anaerobius (6; 2.8%). Seventy-four IDFUs (80%) were infected by multidrug-resistant bacteria, predominantly methicillin-resistant S. aureus and GNB producing extended-spectrum β-lactamases, mainly of the CTX-M variety. Only 4 (3.1%) GNB produced carbapenemases encoded predominantly by bla (VIM). Factors associated with presence of multidrug-resistant bacteria were peripheral neuropathy (adjusted odds ratio [AOR] = 4.05, p = 0.04) and duration of foot infection of more than 1 month (AOR = 7.63, p = 0.02). CONCLUSION: Multidrug-resistant facultative anaerobic bacteria are overrepresented as agents of IDFU. A relatively low proportion of the aetiological agents were anaerobic bacteria.202133824857
142580.9643Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt. Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, bla(OXA-48) and bla(NDM) were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.202439172656
140790.9642World Health Organization priority antimicrobial resistance in Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecium healthcare-associated bloodstream infections in Brazil (ASCENSION): a prospective, multicentre, observational study. BACKGROUND: Carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) are listed by World Health Organization (WHO) as priority antimicrobial-resistant bacteria. Data on WHO Priority Antimicrobial resistance Phenotype (WPAP) bacteria from low- and middle-income countries are scarce. In this study, we investigated the occurrence of WPAP in healthcare-associated bloodstream infections (BSI) in Brazil, an upper-middle-income country in South America. METHODS: ASCENSION was a prospective, multicentre, observational study conducted in 14 hospitals from four of five Brazilian regions. Enterobacterales, A. baumannii, P. aeruginosa, S. aureus and E. faecium BSIs in hospitalised patients were analysed. The primary outcome was the frequency of WPAP among all bacteria of interest. Secondary outcomes were incidence-density of bacteria isolates in hospitalised patients, WPAP proportions within bacterial species, and 28-day mortality. PCR for carbapenemase genes was performed in carbapenem-resistant Gram-negative bacteria. FINDINGS: Between August 15, 2022, and August 14, 2023, 1350 isolates (1220 BSI episodes) were included. WPAP accounted for 38.8% (n = 524; 95% Confidence Interval 32.0-46.1) of all isolates, with CRE (19.3%) as the most frequent, followed by CRAB (9.6%), MRSA (4.9%), VRE (2.7%), and CRPA (2.4%). Incidence-density of all and WPAP isolates were 1.91 and 0.77/1000 patients-day, respectively. Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the most common CRE, corresponding to 14.2% of all BSIs. A. baumannii isolates presented the highest proportion of WPAP (87.8%). Mortality rates were higher in patients with BSIs by WPAP than non-WPAP isolates. KPC (64.4%) was the predominant carbapenemase in CRE, followed by NDM (28.4%) and KPC + NDM co-production (7.1%). OXA-23 was the most frequent in CRAB. INTERPRETATION: A high frequency of WPAP bacteria, particularly CRKP and CRAB, were found in healthcare-associated BSIs in Brazil, posing them as a major public health problem in this country. FUNDING: National Council for Scientific and Technological Development, Brazil.202539957800
2208100.9642Evaluation of the relatedness between the biofilm-associated genes and antimicrobial resistance among Acinetobacter baumannii isolates in the southwest Iran. BACKGROUND AND OBJECTIVES: Increasing antimicrobial resistance among Acinetobacter baumannii (A. baumannii) strains poses a significant challenge, particularly in intensive care units (ICUs) where these bacteria are common causes of hospital infections. Biofilm production is recognized as a key mechanism contributing to this resistance. This study aims to explore the relationship between biofilm production, the presence of biofilm-associated genes, and antibiotic resistance patterns in A. baumannii isolates obtained from ICU patients. MATERIALS AND METHODS: We collected 100 A. baumannii isolates from ICU patients at Nemazee Hospital in Shiraz, Iran. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disk diffusion method, and biofilm production potential was assessed through the tissue culture plate (TCP) method. Additionally, we investigated eleven biofilm-related genes (ompA, bap, csuE, epsA, bla (per-1) , bfmS, pgaB, csgA, fimH, ptk, and kpsMII) in all isolates using polymerase chain reaction (PCR). The REP-PCR technique was utilized to analyze the genetic relatedness of the isolates (Fig. 4). RESULTS: All isolates displayed multi-drug resistance, with the highest resistance rates observed against ceftazidime, cefotaxime, and trimethoprim/sulfamethoxazole (100%). Gentamicin and amikacin showed the lowest resistance rates at 70% and 84%, respectively. A total of 98% of the isolates were capable of biofilm production, with 32% categorized as strong biofilm producers. The most frequently detected biofilm-associated genes included csuE (99%), bfmS (98%), ompA (97%), and pgaB (89%). CONCLUSION: Biofilm production significantly contributes to the prevalence of multi-drug resistant A. baumannii strains. It is essential to implement effective antimicrobial stewardship and develop innovative anti-biofilm strategies to address this global health issue.202540330064
1538110.9641KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: bla(KPC-80), bla(KPC-81), bla(KPC-96) and bla(KPC-97). Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of bla(KPC-2) resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant bla(KPC) genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for bla(KPC-80), bla(KPC-96), and bla(KPC-97) by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including bla(TEM-1), bla(OXA-18) and bla(OXA-1), were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of bla(KPC-2) and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, bla(KPC-2). The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.202438319084
2478120.9641Study on the resistance mechanism via outer membrane protein OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The aim of the present study was to evaluate the imipenem-resistant mechanism via the outer membrane protein (OMP) OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The Pseudomonas aeruginosa was clinically separated and validated by VITEK-2 full-automatic bacteria analyzer. Drug resistance, sensitive antibiotics and minimum inhibitory concentration (MIC) were tested using the drug sensitivity analysis system. The phenotype positive strains of MBL genes were screened using the Kirby-Bauer diffusion method by adding metal ion-chelating agent EDTA on the imipenem susceptibility paper. IMP-1, VIM-1 and SPM metaloenzyme genes were tested by polymerase chain reaction (PCR)-telomeric repeat amplification protocol (TRAP). The OMP OprD2 genes were tested by PCR-TRAP, and the protein expression was tested using western blot analysis. The location of OMP OprD2 was confirmed using the sodium salicylate inhibition test. The results showed that 80 portions (40%) of MBL-positive strains were screened out of 200 specimens. Imipenem-resistant Pseudomonas aeruginosa (IRPA) and MIC values were significantly higher than quality control bacteria and control bacteria (P<0.05). A total of 35 cases with IMP-1 positive, 20 with VIM-1 positive, 16 with SPM positive, 5 with 2 positive genes and 4 with 3 positive genes were screened among MBL positive strains. A total of 150 portions (75%) of OprD2 deficiencies were screened from 200 specimens. The standard strains and sensitive strains showed OprD2 protein bands at 45 kDa while no OprD2 protein bands appeared in OprD2 deficiency strains. It was in accordance with gene detection. In conclusion, OMP OprD2 deficiency and MBL phenotype positivity may be important mechanisms of IRPA.201627882088
2459130.9640In vitro antimicrobial activity and resistance mechanisms of cefiderocol against clinical carbapenem-resistant gram-negative bacteria. BACKGROUND: The rise of carbapenem-resistant gram-negative bacteria (CRGNB) necessitates new therapeutic options such as cefiderocol. OBJECTIVE: To evaluate the in vitro efficacy of cefiderocol against clinical CRGNB and investigate associated resistance mechanisms. METHODS: A total of 370 CRGNB isolates were analyzed. Minimum inhibitory concentration (MIC) values were determined, and whole genome sequencing, efflux pump inhibition assays, and RT-qPCR were conducted to assess resistance-related mutations, gene loss, and expression changes. RESULTS: Cefiderocol demonstrated potent in vitro activity, with high susceptibility rates in C. freundii (100%), K. pneumoniae (93.3%), and E. hormaechei (92.2%), and notable activity against P. aeruginosa (80.0%) and Escherichia coli (76.8%). Efflux pump inhibition by Carbonyl Cyanide m-Chlorophenyl Hydrazone (CCCP) significantly reduced MICs in resistant strains. Key resistance mechanisms included β-lactamase gene variants (bla (OXA-66), bla (OXA-23), bla (SHV-12)), mutations in envZ, cirA, nuoC, ampC, and loss or altered expression of iron transporter genes (piuA, pirA, fepA). CONCLUSION: Cefiderocol is highly effective against CRGNB; however, resistance may arise through diverse mechanisms, including efflux pump activity. Continued surveillance of emerging resistance is essential to guide its optimal clinical use.202541113641
2134140.9639Microbiologic and virulence characteristics of Moraxella catarrhalis isolates from Zambian children presenting with acute pneumonia. BACKGROUND: Moraxella catarrhalis is one of the bacterial pathogens associated with childhood pneumonia, but its clinical importance is not clearly defined. OBJECTIVE: This study aimed to investigate the microbiologic and virulence characteristics of M. catarrhalis isolates obtained from children with pneumonia in Lusaka, Zambia. METHODS: This retrospective, cross-sectional study analyzed 91 M. catarrhalis isolates from induced sputum samples of children less than 5 years of age with pneumonia enrolled in the Pneumonia Etiology Research for Child Health study in Lusaka, Zambia between 2011 and 2014. Bacteria identification and virulence genes detection were performed by PCR and DNA sequencing, while antimicrobial susceptibility testing was determined by the Kirby-Bauer method. RESULTS: All the M. catarrhalis isolates were obtained from good-quality sputum samples and were the predominant bacteria. These isolates harbored virulence genes copB (100%), ompE (69.2%), ompCD (71.4%), uspA1 (92.3%), and uspA2 (69.2%) and were all β-lactamase producers. They showed resistance to ampicillin (100%), amoxicillin (100%), trimethoprim-sulfamethoxazole (92.3%), ciprofloxacin (46.2%), chloramphenicol (45.1%), erythromycin (36.3%), tetracycline (25.3%), cefuroxime (11.0%), and amoxicillin-clavulanate (2.2%), with 71.4% displaying multi-drug resistant phenotype but all susceptible to imipenem (100%). CONCLUSION: This study showed that M. catarrhalis isolates were the predominant or only bacterial isolates from the sputum samples analyzed. The findings provide supportive evidence for the pathogenic potential role of this bacterium in pediatric pneumonia. High multidrug resistance was also observed amongst the isolates, which can result in affected patients not responding to standard treatment, leading to prolonged illness, increased healthcare costs, and risk of death.202236056795
1428150.9638Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt. We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2 years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes bla(KPC), bla(NDM), and bla(OXA-48) using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the bla(NDM) in 28.6% of the isolates and bla(KPC) in 26.8%, bla(NDM) and bla(KPC) were detected together in the same isolate in 5.6%, while bla(OXA-48)-like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.201829936619
2108160.9638Prevalence and Molecular Characterization of Carbapenemase-Producing Multidrug-Resistant Bacteria in Diabetic Foot Ulcer Infections. Background: Diabetic foot ulcers (DFUs) represent severe complications in diabetic patients, often leading to chronic infections and potentially resulting in nontraumatic lower-limb amputations. The increasing incidence of multidrug-resistant (MDR) bacteria in DFUs complicates treatment strategies and worsens patient prognosis. Among these pathogens, carbapenemase-producing pathogens have emerged as particularly concerning owing to their resistance to β-lactam antibiotics, including carbapenems. Methods: This study evaluated the prevalence of MDR bacteria, specifically carbapenemase-producing pathogens, in DFU infections. A total of 200 clinical isolates from DFU patients were analyzed via phenotypic assays, including the modified Hodge test (MHT) and the Carba NP test, alongside molecular techniques to detect carbapenemase-encoding genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48). Results: Among the isolates, 51.7% were confirmed to be carbapenemase producers. The key identified pathogens included Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. The most commonly detected carbapenemase genes were blaKPC (27.6%) and blaNDM (24.1%). Carbapenemase-producing isolates presented high resistance to β-lactam antibiotics, whereas non-carbapenemase-producing isolates presented resistance through mechanisms such as porin loss and efflux pumps. Conclusions: The findings of this study highlight the significant burden of MDR infections, particularly carbapenemase-producing organisms, in DFUs. MDR infections were strongly associated with critical clinical parameters, including pyrexia (p = 0.017), recent antibiotic use (p = 0.003), and the severity of infections. Notably, the need for minor amputations was much higher in MDR cases (p < 0.001), as was the need for major amputations (p < 0.001). MDR infections were also strongly associated with polymicrobial infections (p < 0.001). Furthermore, Wagner ulcer grade ≥II was more common in MDR cases (p = 0.002). These results emphasize the urgent need for enhanced microbiological surveillance and the development of tailored antimicrobial strategies to combat MDR pathogens effectively. Given the high prevalence of carbapenem resistance, there is an immediate need to explore novel therapeutic options to improve clinical outcomes for diabetic patients with DFUs.202539857026
2454170.9638Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms. BACKGROUND: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. RESULTS: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. CONCLUSIONS: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.202134798825
2115180.9637Assessment of carbapenemase genes and antibiotic resistance profiles in ceftazidime-avibactam resistant Klebsiella pneumoniae isolates: A single-center cross-sectional study. BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is an urgent global health threat due to its rapid spread and limited treatment options. Ceftazidime-avibactam exhibits broad efficacy against gram-negative bacteria, including CRKp; however, emerging resistance to this agent is increasingly reported. Understanding the prevalence of ceftazidime-avibactam resistance and the underlying carbapenemase genes is critical for optimizing antimicrobial stewardship and guiding clinical management. This study aimed to determine the prevalence of ceftazidime avibactam resistance among CRKp isolates collected from various clinical specimens, and to analyze their associated carbapenemase genes and antibiotic resistance profiles. METHODS: This cross-sectional study analyzed 312 K pneumoniae isolates obtained from various clinical specimens of hospitalized patients at a tertiary care hospital in Turkey. Antibiotic susceptibility testing was performed using the disk diffusion method for ceftazidime-avibactam and broth microdilution for both colistin and ceftazidime-avibactam. Molecular detection of carbapenemase genes was carried out using polymerase chain reaction. RESULTS: Ceftazidime-avibactam resistance was identified in 21.5% (67/312) of CRKp isolates. Among these isolates, 37.3% harbored both OXA-48 and NDM genes, 13.4% carried NDM alone, 10.4% carried OXA-48 alone, and 38.8% lacked these genes. The majority of resistant isolates originated from urine (31.3%), followed by tracheal aspirate (29.9%), and blood (22.4%) specimens. The prevalence of colistin susceptibility among ceftazidime-avibactam-resistant CRKp isolates was 56.7%. CONCLUSIONS: The coexistence of NDM and OXA-48 genes is a major contributor to ceftazidime-avibactam resistance in CRKp isolates, particularly in urinary and respiratory tract infections. These findings underscore the need for ongoing surveillance and tailored antibiotic stewardship programs to control the spread of resistance in hospital settings.202541088587
2269190.9636Genomic detection of Panton-Valentine Leucocidins encoding genes, virulence factors and distribution of antiseptic resistance determinants among Methicillin-resistant S. aureus isolates from patients attending regional referral hospitals in Tanzania. BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a formidable public scourge causing worldwide mild to severe life-threatening infections. The ability of this strain to swiftly spread, evolve, and acquire resistance genes and virulence factors such as pvl genes has further rendered this strain difficult to treat. Of concern, is a recently recognized ability to resist antiseptic/disinfectant agents used as an essential part of treatment and infection control practices. This study aimed at detecting the presence of pvl genes and determining the distribution of antiseptic resistance genes in Methicillin-resistant Staphylococcus aureus isolates through whole genome sequencing technology. MATERIALS AND METHODS: A descriptive cross-sectional study was conducted across six regional referral hospitals-Dodoma, Songea, Kitete-Kigoma, Morogoro, and Tabora on the mainland, and Mnazi Mmoja from Zanzibar islands counterparts using the archived isolates of Staphylococcus aureus bacteria. The isolates were collected from Inpatients and Outpatients who attended these hospitals from January 2020 to Dec 2021. Bacterial analysis was carried out using classical microbiological techniques and whole genome sequencing (WGS) using the Illumina Nextseq 550 sequencer platform. Several bioinformatic tools were used, KmerFinder 3.2 was used for species identification, MLST 2.0 tool was used for Multilocus Sequence Typing and SCCmecFinder 1.2 was used for SCCmec typing. Virulence genes were detected using virulenceFinder 2.0, while resistance genes were detected by ResFinder 4.1, and phylogenetic relatedness was determined by CSI Phylogeny 1.4 tools. RESULTS: Out of the 80 MRSA isolates analyzed, 11 (14%) were found to harbor LukS-PV and LukF-PV, pvl-encoding genes in their genome; therefore pvl-positive MRSA. The majority (82%) of the MRSA isolates bearing pvl genes were also found to exhibit the antiseptic/disinfectant genes in their genome. Moreover, all (80) sequenced MRSA isolates were found to harbor SCCmec type IV subtype 2B&5. The isolates exhibited 4 different sequence types, ST8, ST88, ST789 and ST121. Notably, the predominant sequence type among the isolates was ST8 72 (90%). CONCLUSION: The notably high rate of antiseptic resistance particularly in the Methicillin-resistant S. aureus strains poses a significant challenge to infection control measures. The fact that some of these virulent strains harbor the LukS-PV and LukF-PV, the pvl encoding genes, highlight the importance of developing effective interventions to combat the spreading of these pathogenic bacterial strains. Certainly, strengthening antimicrobial resistance surveillance and stewardship will ultimately reduce the selection pressure, improve the patient's treatment outcome and public health in Tanzania.202539833938