Antibiotic Susceptibility Research Articles

Ceftazidime-avibactam treatment dilemma of blaKPC−2-containing Klebsiella pneumoniae due to the development of co-existence of mixed strains carrying blaKPC−2 or blaKPC−33 in lung transplant recipients

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant threat to immunocompromised populations, including lung transplant recipients. This study investigates mixed CRKP strains carrying either blaKPC−2 or blaKPC−33 following ceftazidime-avibactam (CAZ/AVI) exposure, particularly in the context of lung transplantation. Mixed CRKP strains with shifting resistance phenotypes were frequently identified in patients exposed to CAZ/AVI. We aimed to elucidate the transitional state of blaKPC variants by selecting CAZ/AVI-sensitive and -resistant CRKP strains from a lung transplantation patient.MethodsThe blaKPC-variant-carrying CRKP strains were collected from lung transplant recipients exposed to CAZ/AVI in less than two years. Antibiotic susceptibility testing (AST) was conducted using microbroth dilution, and whole-genome sequencing (WGS) was used to identify genotypes and resistance mechanisms. Limiting dilution, drop-plate, and in vitro induction experiments determined blaKPC-variant changes during CAZ/AVI administration. qPCR primers/probes were designed to identify blaKPC−2 mutations.ResultsAmong 104 lung transplant recipients infected by blaKPC-harboring CRKP strains and receiving CAZ/AVI, 10 (9.6%) experienced changing resistance phenotypes. The limiting dilution method found that Patient 10’s CRKP strains carried either blaKPC−2 or blaKPC−33. The drop-plate experiment showed differing growth patterns on CAZ/AVI mediums. The in vitro induction experiment demonstrated shifting from blaKPC−2 to blaKPC−33.ConclusionsThe study identified a “transitional state” of the mixed CRKP strains carrying either blaKPC−2 or blaKPC−33 in CAZ/AVI-exposed patients. Molecular diagnostics are crucial for identifying mixed strains and the transitional state of blaKPC variants, guiding treatment decisions in this complex landscape.

Nanomotion technology: an innovative method to study cell metabolism in Escherichia coli, as a potential indicator for tolerance

Introduction. Antibiotic tolerance corresponds to the bacterial ability to survive a transient exposure to antibiotics and is often associated with treatment failure. Current methods of identifying tolerance based on bacterial growth are time-consuming. This study explores the use of a growth-independent method utilizing nanomotion technology to detect antibiotic-tolerant bacteria. Hypothesis. The nanomotion signal obtained from a nanomechanical sensor measures real-time metabolic activity and cellular processes and could provide valuable information about the tolerance of bacteria to antibiotics that cannot be detected by standard antibiotic susceptibility tests. Aim. The aim of this study is to investigate the potential of nanomotion technology to record antibiotic-tolerant bacteria. Methodology. We generated a slow-growing Escherichia coli strain by manipulating mazF expression levels and confirmed its viability by several standard methods. We subsequently measured its nanomotion and the nanomotion of the WT E. coli in the presence or absence of antibiotics. Supervised machine learning was employed to distinguish slow-growing from exponentially growing bacteria. Observations for bacterial nanomotions were confirmed by standard kill curves. Results. We distinguished slow-growing from exponentially growing bacteria using specific features from the nanomotion signal. Furthermore, the exposition of both growth phenotypes to polymyxin decreased the nanomotion signal indicating cell death. Similarly, when exponentially growing cells were exposed to ampicillin, an antibiotic whose efficacy depends on the growth rate, the nanomotion signal also decreased. In contrast, the nanomotion signal remained unchanged for slow-growing bacteria upon exposure to ampicillin. In addition, antibiotic exposure can cause bacterial elongation, in which the biomass of a cell increases without cell division. By overexpressing sulA, we mimicked antibiotic-induced elongation. Differences in the nanomotion signal were observed when comparing elongating and non-elongating phenotypes. Conclusion. This work shows that nanomotion signals entail information about the reaction to antibiotics that standard MIC-based antibiotic susceptibility tests cannot detect. In the future, nanomotion-based antibiotic tolerance tests could be developed for clinical use in chronic or relapsing infections.

Characterization of phage HZY2308 against Acinetobacter baumannii and identification of phage-resistant bacteria

Background Acinetobacter baumannii (AB) is a notable cause of hospital-acquired infections, with carbapenem-resistant Acinetobacter baumannii (CRAB) classified as a high-priority critical pathogen. Bacteriophage therapy is emerging as a promising alternative to combat drug-resistant bacterial infections. In this study, a lytic phage, HZY2308, was isolated from hospital sewage, and the biological properties, biosafety and anti-biofilm properties of phage HZY2308 were characterized and identified. Moreover, the antibacterial effect of phage HZY2308 in combination with antibiotics was investigated, and the apparent characteristics of phage-resistant strain AB48-R were demonstrated, which provided data support for further studies to elucidate the mechanism of generating phage resistance.MethodsPhage HZY2308 was isolated by double agar plate method using clinical strain AB48 as the host bacterium. The morphology of phage HZY2308 was identified by transmission electron microscopy (TEM), and biological characteristics of phage HZY2308 were identified by host range, the efficiency of plating (EOP), sensitivity to temperature, pH, and chloroform, one-step growth curve, the optimal multiplicity of infection (MOI), and detection of endotoxin and cytotoxicity. Besides, the complete genome map of HZY2308 was constructed using CGview, and the phylogenetic tree of HZY2308 was constructed with MEGA. Additionally, the full genomic sequence of phage HZY2308 and the selected phage were compared using Easyfig. Checkerboard test of phage HZY2308 in combination with tigecycline (TGC) was performed to investigate their synergistic effect and bactericidal kinetics. The effect of HZY2308 on biofilm was investigated by semi-quantitative staining of biofilm with crystal violet, determination of bacterial activity in biofilm by 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl) -2 H-tetrazolium-5-carboxanilide (XTT) assay and observation of biofilm structure by fluorescence microscopy. Finally, Phage-resistant bacteria AB48-R were characterized by colony-forming capacity, morphology, growth curves, adsorption efficiency, and antibiotic susceptibility assays.ResultsA lytic phage, HZY2308, was isolated from hospital sewage, which exhibited advantageous traits such as a brief incubation period, large burst size, and robust stability. Safety assessments conducted at both genetic and cellular levels also have yielded positive outcomes. Besides, phage HZY2308 effectively inhibited AB biofilm formation and disrupted established biofilm structures. Furthermore, a synergistic antibacterial effect was noted when phage HZY2308 was combined with tigecycline. Interestingly, the phage-resistant strain, AB48-R was screened through natural selection. Compared to the wild strain AB48, the adsorption efficiency of the phage to AB48-R diminished. However, AB48-R’s sensitivity to antibiotics such as cefepime, gentamicin, amikacin, and tobramycin increased, indicating an evolutionary trade-off.ConclusionsPhage HZY2308 shows strong antimicrobial potential, especially in combination with tigecycline, and the phage-resistant strain exhibits increased antibiotic sensitivity.

Antibiotic susceptibility testing using minimum inhibitory concentration (MIC) assays

Antimicrobial resistance is due to genetic changes that allow bacteria to evade antibiotic treatment. Antimicrobial susceptibility testing is critical for the detection of antibiotic-resistant strains, the selection of effective therapeutic strategies against bacterial infections, and the evaluation of the efficacy of novel antimicrobials. Among the variety of clinical microbiology methods used for antibiotic susceptibility testing, minimum inhibitory concentration (MIC) assays have become the gold standard in clinical practice. MIC assays determine the lowest concentration of an antimicrobial agent that is required to inhibit visible bacterial growth in vitro. Here, we outline MIC assay protocols, in strict accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines that aim to assess the susceptibility of non-fastidious organisms to antimicrobial agents. The protocols described in this methods paper are intended to aid the performance of reliable and informative MIC assays for research purposes that are in line with clinical microbiology practices.

Prevalence of Vancomycin-Resistant Enterococcus spp. Isolates in Southeast Iran, Zahedan

Background: Enterococci are clinically significant, especially in individuals with compromised immune systems, which are recognized as a critical contributor to nosocomial infections. Objectives: This study aimed to determine antibiotic susceptibility profiles and vancomycin-resistance Enterococcus (VRE) isolates in Zahedan, Iran. Methods: A hospital-based cross-sectional study was conducted at Ali Ibne Abi Talib Hospital from March 2019 to February 2022. Clinical samples included urine, blood, abscesses and other body fluids from patients referred to this hospital during the study period. A total of 3000 patients were included in the study, and the Kirby-Bauer method was used to test isolates identified by morphological and biochemical characteristics for antibiotic susceptibility. Data were analyzed using SPSS software version 20. P-values < 0.05 were considered statistically significant. Results: Out of 3000 culture-positive clinical samples comprised of 82 urine, ten blood, one tracheal, two wound, and one pulmonary isolate, were identified as Enterococcus faecalis (69.1%), Enterococcus faecium (7.3%), and Enterococcus spp. (23.6%). Moreover, two-thirds of isolates were multidrug-resistant (MDR). Although 66.7% and 63.9% were sensitive to nitrofurantoin and linezolid, resistance to ciprofloxacin (74.2%) and ampicillin (65.9%) was frequently observed. Nitrofurantoin or linezolid were the only effective antibiotics for UTIs. In addition, 50% of isolates were resistant to vancomycin. Conclusions: Based on the results, a combination of vancomycin/or linezolid and nitrofurantoin for urine infections was effective against Enterococci in this clinical center. Nevertheless, continuous and frequent surveillance for resistance patterns is necessary for the reasonable and evidence-based use of antibiotics.

Avian Pathogenic Escherichia coli Isolated in Poultry Farms in Bangladesh that Use Antibiotics Extensively.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is causing economic losses to the global poultry industry. Increased prevalence of antibiotic resistance in APEC is the leading cause for increased indiscriminate use of various antimicrobial compounds in farms. The study aimed to investigate the presence of phenotypic and genotypic markers for antibiotic resistance, metals, and biocides in APEC from Bangladeshi poultry and details about the antimicrobials used in poultry farms. In total, 55 APEC were isolated from hearts or liver samples of 86 sick or dead chickens using culture on agar plate and biochemical testing. APEC isolates were tested for antibiotic susceptibility to 14 antimicrobial agents according to the European Committee on Antimicrobial Susceptibility Testing. A series of PCRs was performed to screen the presence of genes for quinolones, colistin, aminoglycosides, ESBL, metals, and biocides. Detailed information regarding antibiotic use was collected from farmers during clinical investigations. Resistance was found to 10 antibiotics and prevalence was as follows: ampicillin (86%), ciprofloxacin (86%), trimethoprim-sulphamethoxazole (73%), chloramphenicol (33%), mecillinam (13%), gentamicin (11%), cefoxitin (11%), cefotaxime (9%), tigecycline (2%), and nitrofurantoin (2%). The most common multiresistance phenotype was CIP-AMP-SXT, and 35% of isolates were multidrug resistant. Genotypic analysis confirmed the presence of quinolone resistance genes [qnrS1 and aac-(6')-lb-cr], silver-resistant genes (silE), and mercury-resistant genes (merA) but not others. In total, 88% farmers were using different antimicrobial compounds, and, of them, 56% were using antimicrobials without prescriptions from veterinarians. Ciprofloxacin was most extensively used followed by oxytetracycline. Critically important antibiotics like ciprofloxacin, colistin, and gentamicin are extensively used in the farms. This study confirmed the presence of antibiotics, metals, and biocide-resistant APEC in poultry farms in Bangladesh. Increased resistance to quinolones is a serious ongoing problem. The indiscriminate use of antibiotics in poultry farms is alarming and should be stopped.

Bacteriology and Physicochemical Analysis of Borehole Water in Some Hostels of a Tertiary Institution in Port Harcourt, Nigeria

Several factors influence the quality of water for human consumption. Most of these factors are more of anthropogenic origin than nature and thus exercises an untold health effect on the end-users of such water body. Bacteriology and Physicochemical Analysis of Borehole Water in Some Hostels of a Tertiary Institution in Port Harcourt, Nigeria. Water samples were collected from six (6) hostels for each study period, and subjected to standard laboratory procedures to analyze for parameters such as pH, nitrite, nitrate, dissolved oxygen (DO), electrical conductivity (EC), Phosphate, as well as standard procedures for antibiotics susceptibility and molecular characterization. Result of the physicochemical parameters showed that the parameters ranged from 5.26±3.53 mg/L to 7.61±0.01 mg/L forpH; 0.61±0.01 to 2.56±0.01 mg/L for Dissolved Oxygen (DO); 96±1.41 μS/cm to 503.5±0.71μS/cm for electrical conductivity; 4.56±0.03 mg/L to 20.3±0.01 mg/L for nitrate; 0.003±0mg/L to 0.03±0mg/L for nitrite; 0.12±0mg/L to 0.17±0mg/L for phosphate. Statistical evaluation showed there was a significant difference (p < 0.05) between the mean values of physicochemical parameters studied, except for pH that recorded no statistical difference (p > 0.05). From the study, 5.6% of Staphylococcus species were resistant to Pefloxacin and Rocephin, while 5.9% of Bacillus species were resistant to Ampiclox. Similarly, 3.33% of Salmonella species were resistant to Augmentin, while the rest of the gram negative isolates were either susceptible or within an intermediate range of susceptibility to the antibiotics tested. Molecular characterization of some bacterial species isolated had pairwise identity with Klebsiella pneumonia strain CP27 and Escherichia coli strain RIZHAO 498.1, Staphylococcus haemolyticus strain SH1. The study has shown that the physicochemical water quality variables were all within the WHO limits, with bacterial species having high percentage susceptibility to the antibiotics tested. Regular water quality monitoring is however recommended to ensure continuous access to water of good quality in hostels within the university community.

Pasteurella multocida from deep nasal swabs and tracheobronchial lavage in bovine calves from Sweden

BackgroundBovine respiratory disease (BRD) is common in intensively raised cattle and is often treated with antibiotics. For practitioners, knowledge of the bacteria involved in an outbreak and their antibiotic susceptibility is warranted. To this end, samples from the upper or lower respiratory tract of calves can be submitted for bacteriological culture and susceptibility testing of relevant isolates. However, it is debated whether isolates from the upper respiratory tract are representative of bacteria causing infections in the lower respiratory tract. In this study, we used MALDI-TOF MS, multilocus sequence typing (MLST) and core-genome multilocus sequence typing (cgMLST) to compare culture results of 219 paired samples (sample pairs) of deep nasal swabs (DNS) and tracheobronchial lavage (TBL). The sample pairs came from 171 calves in 30 calf groups across 25 farms with 48 calves sampled twice.ResultsThe predominant bacterial pathogen was Pasteurella multocida, which was isolated from 37.4% of DNS and 22.4% of TBL. There was no statistically significant difference in isolation frequency of P. multocida between calves considered healthy and those suspected for BRD for DNS (P = 0.778) or TBL (P = 0.410). Among the 49 sample pairs where P. multocida was isolated from TBL, the same species was isolated from DNS in 29 sample pairs (59.2%). Isolates from 28 of these sample pairs were evaluated by MLST, and in 24 pairs (86.0%) P. multocida from DNS and TBL were of the same sequence type (ST). Moreover, cgMLST showed that the genetic distance between isolates within 21 of the 28 sample pairs (75.0%), was less than two alleles, and DNS and TBL isolates were considered identical. In seven sample pairs (25%), the genetic distance was greater, and DNS and TBL isolates were considered nonidentical.ConclusionsPasteurella multocida was readily isolated from DNS and in calves where this species was isolated also from TBL, DNS and TBL isolates were identical in 75% of the sample pairs. This suggests that during an outbreak of BRD, submission of DNS samples from 4 to 6 calves could be a convenient approach for practitioners seeking guidance on P. multocida present in the lower respiratory tract and their antibiotic susceptibility.

Cystic fibrosis sputum media induces an overall loss of antibiotic susceptibility in Mycobacterium abscessus

AbstractMycobacterium abscessus complex (MABSC) comprises a group of environmental microorganisms, which are a concerning cause of opportunistic respiratory infections in patients with cystic fibrosis or bronchiectasis. Only 45.6% of MABSC treatments are successful, and therefore this is a need to discover new antimicrobials that can treat these pathogens. However, the transferability of outcomes to the clinic is flawed by an inability to accurately represent the lung environment within the laboratory. Herein, we apply two preestablished formulations of sputum media (ACFS and SCFM1) to MABSC antibiotic susceptibility testing. Using conventional broth microdilution, we have observed strain and antibiotic dependent alterations in antimicrobial sensitivity in each sputum media compared standard laboratory media (7H9), with an overall reduction in susceptibility within the physiologically relevant conditions. We provide a timely contribution to the field of M. abscessus antibiotic discovery by emphasising the need for improved physiological relevance.

Characterization of Extended Spectrum Beta-lactamase Uropathogens Isolated from Refugees with Urinary Tract Infections in Nakivale Refugee Settlement Camp, Southwestern Uganda

The World Health Organization estimates one in four individuals has had at least one urinary tract infection (UTI) episode requiring treatment with an antimicrobial agent. At Nakivale refugee camp, the overwhelming number of refugees often associated with poor living conditions predispose the refugees to urinary tract infections. This study determined the prevalence of UTIs, the antimicrobial susceptibility pattern of the isolated bacterial pathogens, the prevalence of Extended-Spectrum Beta-Lactamase (ESBL) bacteria, and the molecular characterization of genes encoding ESBLs among refugees in the Nakivale refugee settlement. This was a cross-sectional study that involved 216 outpatients who visited Nakivale Health Centre III between July and September 2020. The urine samples were received and examined at the microbiology laboratory of Mbarara University of Science and Technology. The urine samples were cultured and identified. Antibiotic susceptibility was carried out following CLSI recommended guidelines while the presence of genes encoding ESBL was detected using conventional PCR amplification. The prevalence of UTI was 24.1% (52/216). Staphylococcus aureus was the most prevalent causative agent, accounting for 22/52 (42.31%) of total isolates, followed by Escherichia coli 21/52(40.38%). Multidrug-resistant isolates accounted for 71.15% (37/52). A total of twenty-one isolates (70.0%) were extended spectrum beta-lactamase producing bacteria. The most prevalent genes were TEM beta-lactamase (blaTEM) and CTX-M beta-lactamase (blaCTX-M). The prevalence of UTI among refugees in the Nakivale settlement was high which calls for continuous epidemiological surveys to determine the prevalence of multi-drug resistance uropathogens including ESBL-producing organisms across refugee camps in Uganda.

Pulsed electric field at resonance frequency combat Klebsiella pneumoniabiofilms.

Healtcare-associated infections have increased due to the development of antimicrobial resistance (AMR) of Gram-negative pathogens (GNPs) and the development of outbreacks over the past two decades. In this work, we investigated how exposure to positive electric pulses affects the growth characteristics of Klebsiella pneumonia (K. pneumonia), a common cause of pneumonia. We explored the impact of varying exposure frequencies (0.2-2Hz) and time (15-90min, at resonance frequency) on bioelectric signals produced during cell division, biofilm formation, and bacterial antibiotic susceptibility. Our research found that an extremely low-frequency pulsed electric field (ELF-PEF) significantly inhibited K. pneumonia growth. Specifically, exposure to 0.8Hz for one hour increased the antibiotic susceptibility of K. pneumonia to inhibitors of cell wall formation, proteins, β-lactamase, DNA, and other substances. We also noticed a notable decrease in K. pneumonia biofilm development exposed to ELF-PEF. Our results suggest that the interaction of K. pneumonia cells with ELF-PEF at the specified frequency and time alters cellular activity and bacterial structure. This technique may be used in the future to treat K. pneumonia infections both in vitro and in vivo.

Comprehensive genomic analysis and evaluation of in vivo and in vitro safety of Heyndrickxia coagulans BC99

This study aimed to evaluate the safety profile and beneficial effects of Heyndrickxia coagulans strain BC99 (BC99) for potential use in functional foods and pharmaceuticals. We began with whole genome sequencing of BC99, followed by a comprehensive safety assessment comprising genome analysis, hemolysis, cytotoxicity, antibiotic susceptibility tests, and cell adhesion and tolerance studies, along with acute and subacute oral toxicity studies in animal models. BC99 was isolated from a well-characterized collection originating from the feces of a healthy infant. Our results indicated no hemolytic activity on Columbia blood agar plates and broad antibiotic sensitivity, including to gentamicin, ampicillin, chloramphenicol, ciprofloxacin, and others. Cytotoxicity testing confirmed no adverse effects on HT-29 cells and significant adhesive properties to intestinal epithelial cells. Tolerance tests demonstrated over 90% viability of BC99 under simulated gastrointestinal conditions. In vivo studies in mice and rats confirmed the absence of adverse effects following oral administration. Collectively, these findings support BC99’s robust tolerance to gastrointestinal environments, strong adhesion capabilities, and a broad spectrum of antibiotic resistance, underlining its potential as a safe and effective agent for gut microbiota modulation and host health enhancement.

Bacterial isolates and antibiotic sensitivity in canine bacterial keratitis in Korea.

To analyze bacterial isolates associated with canine bacterial keratitis and their antibiotic susceptibility patterns in Korea, focusing on multidrug resistance (MDR) and identifying effective antibiotic combinations for clinical treatment. A total of 146 dogs diagnosed with suspected bacterial keratitis between October 2022 and October 2023 in Korea, with 157 eye samples collected for analysis. Eye samples were cultured to isolate bacteria, and antibiotic susceptibility testing was performed using the minimum inhibitory concentration (MIC) method. Bacterial identification was conducted using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF). The study assessed the efficacy of individual antibiotics and combination therapies. Bacteria were isolated in 55.4% of the samples. The most common genera were Staphylococcus species (48.5%, 48/99), Streptococcus species (13.1%, 13/99), Pseudomonas species (9.1%, 9/99), and Escherichia coli (9.1%, 9/99). Amikacin (84.8%) showed the highest antibiotic susceptibility, while doxycycline exhibited the lowest (17.2%). The most effective antibiotic combinations were amikacin-moxifloxacin (93%). MDR isolates accounted for 52.5% (52/99) of the total bacterial samples. Staphylococcus species were the most common isolates, with 52.5% showing MDR, underscoring the need to curb antibiotic misuse. While antibiotics like amikacin demonstrated high susceptibility rates, their use should be reserved for resistant infections to prevent further resistance development. Rather than focusing solely on finding effective combinations of antibiotics, it is crucial to consider alternative treatment strategies that offer more sustainable solutions. Rather than relying on antibiotic combinations, attention should shift to sustainable alternatives to treat bacterial keratitis and reduce antibiotic dependence in clinical practice.

Evaluation of a Burkholderia ambifaria strain from plants as a novel promising probiotic in dental caries management

ABSTRACT Background Probiotics serve as a novel preventive or therapeutic approach for dental caries owing to their ability to reverse dysbiosis and restore a healthy microbiota. Here, we identified Burkholderia ambifaria AFS098024 as a probiotic candidate isolated from plants. Methods The safety of B. ambifaria was evaluated by hemolytic activity, D-lactic acid production and antibiotic susceptibility. In vitro biofilm model derived from the saliva of caries-free and caries-active donors and in vivo rat caries model were used to assess the efficacy of B. ambifaria in caries prevention and treatment. Results B. ambifaria was safe as a probiotic candidate and it could integrate with in vitro biofilm model. It significantly reduced the biomass and lactate production of biofilms from caries-active donors and disrupted biofilm structures. B. ambifaria effectively reduced the severity of carious lesions in rat molars, regardless of the inoculation sequence. Molars pretreated or treated with B. ambifaria demonstrated notably higher enamel volumes. Additionally, colonization of rat molars by B. ambifaria persisted for 6 weeks. Conclusion The B. ambifaria strain used in this study holds promise as a probiotic for inhibiting dental caries, both in vitro and in vivo.

Causative Agents and Antibiotic Resistance in Nosocomial Sepsis: A Cross-Sectional Multicenter Study from Isfahan Province, Iran

Background: Nosocomial sepsis is a leading cause of morbidity and mortality worldwide. Understanding the antibiotic susceptibility patterns of pathogens specific to each geographic region is essential for effective patient management. Methods: This cross-sectional study aimed to investigate the microorganisms causing nosocomial sepsis and their antibiotic sensitivity in patients admitted to three referral hospitals in Isfahan, Iran. Clinical information and blood culture results were obtained from hospital records. Results from patients without signs of sepsis, as well as contaminants and community-acquired isolates, were excluded. Stratification was conducted based on sex and age (< 20 and > 20 years) categories. Results: In this study, 267 patients with hospital-acquired sepsis were identified, with gram-negative bacteria accounting for 77.2% of infections. The most common pathogens included Klebsiella spp. (35.2%), Acinetobacter spp. (21.7%), Enterococcus spp. (13.5%), Escherichiacoli (8.6%), Pseudomonasaeruginosa (8.6%), and Staphylococcusaureus (8.2%). The highest resistance among gram-negative bacteria was observed with cefepime (80.3%), ceftazidime (76.5%), cefotaxime/ceftriaxone (70.0%), trimethoprim-sulfamethoxazole (68.6%), ciprofloxacin (68.2%), meropenem (60.0%), and amikacin (50.3%). All gram-negative isolates were sensitive to colistin. Among gram-positive bacteria, the highest resistance was to ciprofloxacin (77.3%), ampicillin (75.7%), clindamycin (74.4%), vancomycin (64.9%), and gentamicin (30.0%). All gram-positive isolates in this study were sensitive to linezolid. Conclusions: The study indicates a high level of resistance among bacterial agents causing nosocomial sepsis in the studied area. Consequently, treatment may necessitate the use of last-line antibiotics, such as linezolid and colistin.

Listeria monocytogenes in Fruits and Vegetables: Antimicrobial Resistance, Biofilm, and Genomic Insights

Background/Objectives: Listeria monocytogenes is a foodborne pathogen that can infect both humans and animals and cause noninvasive gastrointestinal listeriosis or invasive listeriosis. The objectives of this study were to determine the genetic diversity of L. monocytogenes; the genes associated with its resistance to antibiotics, benzalkonium chloride (BC), and cadmium chloride (CdCl2); and its biofilm formation. Methods: A total of 132 fresh fruits (44 samples) and vegetables (88 samples) were selected for this study. The genetic diversity of the isolates and the genes associated with their antibiotic resistance were determined using PCR amplification; meanwhile, their levels of susceptibility to antibiotics were determined using the agar diffusion method. Their levels of resistance to BC and CdCl2 were determined using the minimum inhibitory concentration method, and their capacity for biofilm formation was evaluated using the crystal violet staining method. Results: A total of 17 L. monocytogenes strains were collected: 12.8% (17/132) from fresh fruits and vegetables in this study. The isolates of L. monocytogenes belonged to phylogenetic groups I.1 (29.4% (5/17); serotype 1/2a) and II.2 (70.5% (12/17); serotype 1/2b); strains containing Listeria pathogenicity islands (LIPIs) were also identified at prevalence rates of 100% for LIPI-1 and LIPI-2 (17/17), 29.4% for LIPI-3 (5/17), and 11.7% for LIPI-4 (2/17). The antibiotic susceptibility tests showed that the L. monocytogenes isolates exhibited six different multiresistant patterns, with multiple antibiotic resistance (MAR) index of ≥0.46 (70.5%; 12/17); additionally, the genes Ide, tetM, and msrA, associated with efflux pump Lde, tetracycline, and ciprofloxacin resistance, were detected at 52.9% (9/17), 29.4% (5/17), and 17.6% (3/17), respectively. The phenotypic tests showed that 58.8% (10/17) of cadmium-resistant L. monocytogenes isolates had a co-resistance of 23.5% (4/17) to BC. Finally, all strains of L. monocytogenes exhibited moderate biofilm production. Conclusions: The results of this study contribute to our understanding of the persistence and genetic diversity of L. monocytogenes strains isolated from fresh fruits and vegetables; in addition, their resistance to CdCl2, which is correlated with co-resistance to BC disinfectant, is helpful for the food industry.

Comparison of Carbapenemases and Extended-Spectrum β-Lactamases and Resistance Phenotypes in Hospital- and Community-Acquired Isolates of Klebsiella pneumoniae from Croatia

K. pneumoniae harbors various antibiotic resistance determinants like extended-spectrum and plasmid-mediated AmpC β-lactamases and carbapenemases. In the last three years, in the period of intense population aging, migrations and climate changes in Europe and Croatia as well, we observed changes in antibiotic resistance patters of carbapenem-resistant K. pneumoniae (CRKP) isolates obtained routinely in community and inpatient setting. The aim was to compare and subsequently analyze CRKP hospital and community isolates resistance mechanisms, traits and molecular epidemiology, in order to analyze the dynamic of resistance trends, carbapenemase types and plasmid epidemiology. Disk diffusion and broth dilution method were the methods of choice to determine antibiotic susceptibility. β-lactamases were screened by phenotypic methods and confirmed with PCR. In total 113 isolates were analysed. Resistance to amoxicillin-clavulanate and ertapenem was confirmed in all strains. High resistance rates (over 90%) were observed for extended-spectrum cephalosporins, and ciprofloxacin. OKNV (OXA-48, KPC, NDM, VIM) testing and PCR detected OXA-48 in 106, NDM in 7 and KPC in only one isolate. ESBLs accompanied carbapenemases in 103 isolates. IncL, associated with OXA-48, was the dominant plasmid type. No significant differences in the resistance profile and resistance determinants were found between hospital and community isolates plasmid type.

Molecular Identification, Histopathology and Antibiotic Susceptibility Profiling of Aeromonas veronii Isolated from Oreochromis niloticus in Bangladesh.

Tilapia (Oreochromis niloticus) is the most widely cultured freshwater fish species in Bangladesh and worldwide. However, commercial tilapia culture systems face increasing challenges from bacterial infections. The objective of this study was to identify the bacterial isolates from infected tilapia inan intensive cage culture farm located along the Shitalakshya River in Bangladesh. Infected fish samples were collected and underwentcomprehensive clinical and post-mortem investigations, followed by phenotypic, biochemical and molecular identification of the bacterial isolates, as well as histopathological and antibiotic susceptibility examinations. Phenotypic and biochemical characterization showed similarities of the -collected isolates with Aeromonas veronii. Moreover, molecular analysis of the bacterial conserved region 16S rRNA also confirmed these isolates as A. veronii. The analysed 16S rRNA sequence (GenBank accession no. PP832815) showed a close relationship (100% identity) with A. veronii from China (GenBank accession no. MT071624) in the NCBI BLAST search, and in the phylogenetic tree, they grouped in a single clade. This close genetic relationship is also supported by the low genetic distance between the isolates. Histopathological analysis revealed gross pathological changes like necrosis, hypertrophy and inflammation in muscle tissues. The isolates were found to be sensitive to multiple antibiotics but resistant to trimethoprim and sulphamethoxazole. This study investigated the presence of A. veronii infection in tilapia (O. niloticus) in an intensive cage culture farm in Bangladesh.

Screening of bile salt hydrolase-producing lactic acid bacteria and evaluation of cholesterol-lowering activity in vitro

Hypercholesterolemia is a great threat to humans. Bile salt hydrolase (BSH)-producing lactic acid bacteria (LAB) may alleviate it, but current screening methods are inadequate. This study aims to establish a more comprehensive and reliable in vitro method to screen BSH-producing LAB with greater potential for anti-hypercholesterolemia. Forty-one LAB were initially isolated from infant feces and 37 strains could produce BSH. Strains with BSH over 2 U/mg for sodium glycodeoxycholate and sodium taurodeoxycholate were used to analyse cholesterol degradation rate, hydrophobicity, self-aggregation, gastric fluid tolerance, and intestinal fluid tolerance. Based on the results of these indicators, Lactococcus lactis Y17, L. rhamnosus Q2, and L. fermentum Q11 with the top scores were considered anti-hypercholesterolemia candidates using a principal component analysis evaluation. Antioxidant and Caco-2 adhesion assays showed that these three strains exhibited excellent antioxidant ability and Caco-2 adhesion ability. Their total antioxidant capacity was above 0.99 μmol/L, while the Caco-2 adhesion rate was higher than 8.22%. Safety assessments, via antibiotic susceptibility and hemolysis tests, confirmed their safety. In the HepG2 cells assay, the TC content of the Y17, Q2, and Q11 groups was reduced by 0.525 mmol/L, 0.426 mmol/L, and 0.581 mmol/L, which verified the potential of the three LAB in anti-hypercholesterolemia. In addition, they could enhance cholesterol uptake and bile acid synthesis in HepG2 cells by upregulating the mRNA expression of bile acid synthase (CYP7A1 and CYP8B1) and cholesterol uptake receptors (NPC1L1 and LDLR). The results showed that the three LAB could be developed into anti-hypercholesterolemia foods with beneficial properties.

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

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