Removal of ceftriaxone and cefotaxime antibiotics from industrial wastewater via complexation with Cu(II).

A high-performance liquid chromatographic method (HPLC) with UV detection is described for determination of ceftriaxone sodium (CFX) and cefotaxime sodium (CFM) content in pharmaceutical industrial wastewater. These methods are based on the detection of these antibiotics via the formation of chelate complexes with Cu(II). The developed Liquid Chromatographic method offers symmetric peak shape, good resolution and reasonable retention time for both drugs. The removal percentage reached about 100 and 92.1% at pH 7.2 for CFX and CFM, respectively. In UV detection, the removal of the chelating antibiotics were based on forming of chelate complexes with Cu(II) which detected at λmax = 253 and 244 nm for CFX and CFM, respectively. Linearity, accuracy and precision were found to be acceptable over the concentration range of 5.99-59.86 µg mL-1 for CFX and 14.33-71.63 µg mL-1 for CFM. The proposed method can be used for the quality control of industrial wastewater containing CFX and CFM.

Lysine and citric acid based pegylated polymeric dendritic nano drug delivery carrier and their bioactivity evaluation.

The main objective of this work is to synthesize multifunctional nanodendritic structural molecules that can effectively encapsulate hydrophilic as well as hydrophobic therapeutic agents. Four different types of fourth-generation lysine-citric acid based dendrimer have been synthesized in this work: PE-MC-Lys-CA-PEG, TMP-MC-Lys-CA-PEG, PE-MS-Lys-CA-PEG, and TMP-MS-Lys-CA-PEG. The antibacterial drug cefotaxime (CFTX) was further conjugated to these dendrimers. The dendrimer and drug-dendrimer conjugate structures were characterized with the help of FTIR,1H-NMR, and 13C-NMR spectroscopy. Zeta sizer, AFM, and HR-TEM techniques were used to investigate the particle size, surface topography, and structural characteristics of drug-dendrimer conjugates. In vitro drug release was then investigated using dialysis method. Various kinetic drug release models were examined to evaluate the type of kinetic drug release mechanism of the formulations. Cytotoxicity study revealed that the dendrimers encapsulated with CFTX exhibited 2-3% toxicity against healthy epithelial cells, indicating their safe use. Plain dendrimers show 10-15% hemolytic toxicity against red blood cells (RBC), and the toxicity was reduced to 2-3% when CFTX was conjugated to the same dendrimers. The 3rd and 4th generation synthesized drug-dendrimer conjugates exhibit a significantly effective zone of inhibition (ZOI) against both Gram-positive and Gram-negative bacteria. For Gram-positive bacteria, the lower concentration of 0.1 mg/mL showed more than 98% inhibition of drug-dendrimer conjugate samples against B. subtilis and more than 50% inhibition against S. aureus using 0.2 mg/mL, respectively. Moreover, samples with concentrations of 0.5 and 1.0 mg/mL exhibited more than 50% inhibition against S. typhimurium and E. coli, respectively.

Transmission of human-pet antibiotic resistance via aerosols in pet hospitals of Changchun.

In recent years, aerosols have been recognized as a prominent medium for the transmission of antibiotic-resistant bacteria and genes. Among these, particles with a particle size of 2 µm (PM2.5) can directly penetrate the alveoli. However, the presence of antibiotic-resistant genes in aerosols from pet hospitals and the potential risks posed by antibiotic-resistant bacteria in these aerosols to humans and animals need to be investigated. In this study, cefotaxime-resistant bacteria were collected from 5 representative pet hospitals in Changchun using a Six-Stage Andersen Cascade Impactor. The distribution of bacteria in each stage was analyzed, and bacteria from stage 5 and 6 were isolated and identified. Minimal inhibitory concentrations of isolates against 12 antimicrobials were determined using broth microdilution method. Quantitative Polymerase Chain Reaction was employed to detect resistance genes and mobile genetic elements that could facilitate resistance spread. The results indicated that ARBs were enriched in stage 5 (1.1-2.1 µm) and stage 3 (3.3-4.7 µm) of the sampler. A total of 159 isolates were collected from stage 5 and 6. Among these isolates, the genera Enterococcus spp. (51%), Staphylococcus spp. (19%), and Bacillus spp. (14%) were the most prevalent. The isolates exhibited the highest resistance to tetracycline and the lowest resistance to cefquinome. Furthermore, 56 (73%) isolates were multidrug-resistant. Quantitative PCR revealed the expression of 165 genes in these isolates, with mobile genetic elements showing the highest expression levels. In conclusion, PM2.5 from pet hospitals harbor a significant number of antibiotic-resistant bacteria and carry mobile genetic elements, posing a potential risk for alveolar infections and the dissemination of antibiotic resistance genes.

Cefotaxime Exposure-Caused Oxidative Stress, Intestinal Damage and Gut Microbial Disruption in Artemia sinica.

Cefotaxime (CTX) is an easily detectable antibiotic pollutant in the water environment, but little is known about its toxic effects on aquatic invertebrates, especially on the intestine. Here, we determined the oxidative stress conditions of A. sinica under CTX exposure with five concentrations (0, 0.001, 0.01, 0.1 and 1 mg/L) for 14 days. After that, we focused on changes in intestinal tissue morphology and gut microbiota in A. sinica caused by CTX exposure at 0.01 mg/L. We found malondialdehyde (MDA) was elevated in CTX treatment groups, suggesting the obvious antibiotic-induced oxidative stress. We also found CTX exposure at 0.01 mg/L decreased the villus height and muscularis thickness in gut tissue. The 16S rRNA gene analysis indicated that CTX exposure reshaped the gut microbiota diversity and community composition. Proteobacteria, Actinobacteriota and Bacteroidota were the most widely represented phyla in A. sinica gut. The exposure to CTX led to the absence of Verrucomicrobia in dominant phyla and an increase in Bacteroidota abundance. At the genus level, eleven genera with an abundance greater than 0.1% exhibited statistically significant differences among groups. Furthermore, changes in gut microbiota composition were accompanied by modifications in gut microbiota functions, with an up-regulation in amino acid and drug metabolism functions and a down-regulation in xenobiotic biodegradation and lipid metabolism-related functions under CTX exposure. Overall, our study enhances our understanding of the intestinal damage and microbiota disorder caused by the cefotaxime pollutant in aquatic invertebrates, which would provide guidance for healthy aquaculture.

Dosing Regimen for Cefotaxime Should Be Adapted to the Stage of Renal Dysfunction in Critically Ill Adult Patients-A Retrospective Study.

Cefotaxime administration is recommended in doses of 3-12 g/day in adults with a Glomerular Filtration Rate (GFR) > 5 mL/min. This study aimed to assess the impact of renal function and obesity on cefotaxime concentrations in intensive care unit (ICU) patients. A retrospective cohort study was conducted on consecutive ICU patients receiving continuous cefotaxime infusion between 2020 and 2022 [IRBN992021/CHUSTE]. Doses were not constant; consequently, a concentration-to-dose ratio (C/D) was considered. Statistical analysis was performed to assess the relationship between cefotaxime concentrations, renal function, and obesity. A total of 70 patients, median age 61 years, were included, with no significant difference in cefotaxime concentrations between obese and non-obese patients. However, concentrations varied significantly by GFR, with underdosing prevalent in patients with normal to increased renal function and overdosing in those with severely impaired renal function. Adjustment of cefotaxime dosing according to GFR was associated with improved target attainment. Cefotaxime dosing in critically ill patients should consider renal function, with higher initial doses required in patients with normal to increased GFR and lower doses in those with severely impaired renal function. Therapeutic drug monitoring may aid in optimising dosing regimens. Prospective studies are warranted to validate these findings and inform clinical practice.

Physiologically-Based Pharmacokinetic Modeling and Dosing Optimization of Cefotaxime in Preterm and Term Neonates.

BACKGROUND: Cefotaxime is commonly used in treating bacterial infections in neonates. To characterize the pharmacokinetic process in neonates and evaluate different recommended dosing schedules of cefotaxime, a physiologically-based pharmacokinetic (PBPK) model of cefotaxime was established in adults and scaled to neonates. METHODS: A whole-body PBPK model was built in PK-SIM® software. Three elimination pathways are composed of enzymatic metabolism in the liver, passive filtration through glomerulus, and active tubular secretion mediated by renal transporters. The ontogeny information was applied to account for age-related changes in cefotaxime pharmacokinetics. The established models were verified with realistic clinical data in adults and pediatric populations. Simulations in neonates were conducted and 100 % of the dosing interval where the unbound concentration in plasma was above the minimum inhibitory concentration (fT>MIC) was selected as the target index for dosing regimen evaluation. RESULTS: The developed PBPK models successfully described the pharmacokinetic process of cefotaxime in adults and were scaled to the pediatric population. Good verification results were achieved in both adults' and neonates' PBPK models, indicating a good predictive performance. The optimal dosage regimen of cefotaxime was proposed according to the postnatal age (PNA) and gestational age (GA) of neonates. For preterm neonates (GA < 36 weeks), dosages of 25 mg/kg every 8 h in PNA 0-6 days and 25 mg/kg every 6 h in PNA 7-28 days were suggested. For term neonates (GA ≥ 36 weeks), dosages of 33 mg/kg every 8 h in PNA 0-6 days and 33 mg/kg every 6 h in PNA 7-28 days were recommended. CONCLUSIONS: Our study may provide useful experience in practicing PBPK model-informed precision dosing in the pediatric population.

In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development.

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.

Chlorogenic acid inhibits macrophage PANoptosis induced by cefotaxime-resistant Escherichia coli.

Antibiotics are commonly used in clinical practice to treat bacterial infections. Due to the abuse of antibiotics, the emergence of drug-resistant strains, such as cefotaxime sodium-resistant Escherichia coli (CSR-EC), has aggravated the treatment of diseases caused by bacterial infections in the clinic. Therefore, discovering new drug candidates with unique mechanisms of action is imperative. Chlorogenic acid (CGA) is an active component of Yinhua Pinggan Granule, which has antioxidant and anti-inflammatory effects. We chose the CGA to explore its effects on PANoptosis in cultured macrophages infected with CSR-EC. In this study, we explored the protective impact of CGA on macrophage cell damage generated by CSR-EC infection and the potential molecular mechanistic consequences of post-infection therapy with CGA on the PANoptosis pathway. Our findings demonstrated that during CSR-EC-induced macrophage infection, CGA dramatically increased cell survival. CGA can inhibit pro-inflammatory cytokine expression of IL-1ß, IL-18, TNF-α, and IL-6. CGA decreased ROS generation and increased Nrf-2 expression at the gene and protein levels to lessen the cell damage and death brought on by CSR-EC infection. Additionally, we discovered that the proteins Caspase-3, Caspase-7, Caspase-8, Caspase-1, GSDMD, NLRP-3, RIPK-3, and MLKL were all inhibited by CGA. In summary, our research suggests that CGA is a contender for reducing lesions brought on by CSR-EC infections and that it can work in concert with antibiotics to treat CSR-EC infections clinically. However, further research on its mechanism of action is still needed.

Efficacy of delafloxacin alone and in combination with cefotaxime against cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae.

OBJECTIVE: We assessed the in vitro activity of delafloxacin and the synergy between cefotaxime and delafloxacin among cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae (CNSSP). METHODS: A total of 30 CNSSP (cefotaxime MIC > 0.5 mg/L) were studied. Serotyping was performed by the Pneumotest-Latex and Quellung reaction. Minimum inhibitory concentrations (MICs) of delafloxacin, levofloxacin, penicillin, cefotaxime, erythromycin and vancomycin were determined by gradient diffusion strips (GDS). Synergistic activity of delafloxacin plus cefotaxime against clinical S. pneumoniae isolates was evaluated by the GDS cross method. RESULTS: Delafloxacin showed a higher pneumococcal activity than its comparator levofloxacin (MIC50, 0.004 versus 0.75 mg/L and MIC90, 0.047 versus >32 mg/L). Resistance to delafloxacin was identified in 7/30 (23.3%) isolates, belonging to serotypes 14 and 9V. Synergy between delafloxacin and cefotaxime was detected in 2 strains (serotypes 19A and 9V). Antagonism was not observed. Addition of delafloxacin increased the activity of cefotaxime in all isolates. Delafloxacin susceptibility was restored in 5/7 (71.4%) strains. CONCLUSIONS: CNSSP showed a susceptibility to delafloxacin of 76.7%. Synergistic interactions between delafloxacin and cefotaxime were observed in vitro among CNSSP by GDS cross method.

Divergent genetic landscapes drive lower levels of AmpC induction and stable de-repression in Serratia marcescens compared to Enterobacter cloacae.

The chromosomally encoded AmpC beta-lactamase is widely distributed throughout the Enterobacterales. When expressed at high levels through transient induction or stable de-repression, resistance to ceftriaxone, a commonly used antibiotic, can develop. Recent clinical guidance suggests, based on limited evidence, that resistance may be less likely to develop in Serratia marcescens compared to the better-studied Enterobacter cloacae and recommends that ceftriaxone may be used if the clinical isolate tests susceptible. We sought to generate additional data relevant to this recommendation. AmpC de-repression occurs predominantly because of mutation in the ampD peptidoglycan amidohydrolase. We find that, in contrast to E. cloacae, where deletion of ampD results in high-level ceftriaxone resistance (with ceftriaxone MIC = 96 µg/mL), in S. marcescens deletion of two amidohydrolases (ampD and amiD2) is necessary for AmpC de-repression, and the resulting ceftriaxone MIC is 1 µg/mL. Two mechanisms for this difference were identified. We find both a higher relative increase in ampC transcript level in E. cloacae ΔampD compared to S. marcescens ΔampDΔamiD2, as well as higher in vivo efficiency of ceftriaxone hydrolysis by the E. cloacae AmpC enzyme compared to the S. marcescens AmpC enzyme. We also observed higher relative levels of transient AmpC induction in E. cloacae vs S. marcescens when exposed to ceftriaxone. In time-kill curves, this difference translates into the survival of E. cloacae but not S. marcescens at clinically relevant ceftriaxone concentrations. In summary, our findings can explain the decreased propensity for on-treatment ceftriaxone resistance development in S. marcescens, thereby supporting recently issued clinical guidance.

Unveiling the Role of Nonionic Surfactants in Enhancing Cefotaxime Drug Solubility: A UV-Visible Spectroscopic Investigation in Single and Mixed Micellar Formulations.

This study reports the interfacial phenomenon of cefotaxime in combination with nonionic surfactants, Triton X-100 (TX-100) and Tween-80 (TW-80), and their mixed micellar formulations. Cefotaxime was enclosed in a micellar system to improve its solubility and effectiveness. TX-100 and TW-80 were used in an amphiphilic self-assembly process to create the micellar formulation. The effect of the addition of TX-100, a nonionic surfactant, on the ability of TW-80 to solubilize the drug was examined. The values of the critical micelle concentration (CMC) were determined via UV-Visible spectroscopy. Gibbs free energies (ΔGp and ΔGb), the partition coefficient (Kx), and the binding constant (Kb) were also computed. In a single micellar system, the partition coefficient (Kx) was found to be 33.78 × 106 and 2.78 × 106 in the presence of TX-100 and TW-80, respectively. In a mixed micellar system, the value of the partition coefficient for the CEF/TW-80 system is maximum (5.48 × 106) in the presence of 0.0019 mM of TX-100, which shows that TX-100 significantly enhances the solubilizing power of micelles. It has been demonstrated that these surfactants are effective in enhancing the solubility and bioavailability of therapeutic compounds. This study elaborates on the physicochemical characteristics and solubilization of reactive drugs in single and mixed micellar media. This investigation, conducted in the presence of surfactants, shows a large contribution to the binding process via both hydrogen bonding and hydrophobic interactions.

Comparing the Frequency of Culture-Positive Late Onset Sepsis With the Use of Ceftazidime Versus Cefotaxime in the NICU.

OBJECTIVE: As broader spectrum antibiotics have been associated with adverse effects, our study evaluated whether the frequency of culture-positive late-onset sepsis (LOS) and multidrug resistant (MDR) infections were increased with the use of ceftazidime as compared with cefotaxime in the neonatal intensive care unit (NICU). METHODS: This was a multihospital, retrospective chart review of patients who received at least 24 hours of ceftazidime or cefotaxime in the NICU between December 1, 2012 and August 31, 2021. Patients were excluded from analysis if they expired during the admission, had an incomplete history, positive cultures for an MDR infection prior to receiving either antibiotic, or received the alternate antibiotic within the same treatment course. RESULTS: A total of 334 patients were included for analysis (ceftazidime, n = 147; cefotaxime, n = 187). The average birth weight was lower in the ceftazidime cohort compared with the cefotaxime cohort [1.46 kg (95% CI, 1.29-1.63 kg) versus 1.93 kg (95% CI, 1.75-2.11 kg), p = 0.0002] with a corresponding lower gestational age [28.9 weeks (95% CI, 28.0-29.9 weeks) versus 31.7 weeks (95% CI, 30.8-32.6 weeks), p = 0.0001]. Adjusting for baseline differences showed a protective effect for ceftazidime (OR = 0.32; 95% CI, 0.16-0.62; p = 0.0009). There was no statistically significant difference in the frequency of MDR infections between the cohorts (OR = 0.25; 95% CI, 0.053-1.14; p = 0.07), however this study was underpowered to detect the difference noted. CONCLUSIONS: Ceftazidime appears to be a safe and effective alternative treatment option compared with cefotaxime in the NICU with no increase in the risk of culture-positive LOS or MDR infections.

Haemophilus influenzae blood-stream infection and third-generation cephalosporin susceptibility testing: a comparative case study using EUCAST and CLSI guidelines.

Introduction: In this comparative case study, we discuss clinically relevant discrepancies of antimicrobial susceptibility testing (AST) interpretation for ceftriaxone against a non-typable, beta-lactamase negative, ampicillin-resistant (BLNAR) Haemophilus influenzae isolated from a blood culture. Case report: A 74-year-old man presented with a 3 day illness characterized by shortness of breath and dry cough, and was noted to be febrile and hypoxic on admission. A blood culture bottle flagged positive with Gram-negative coccobacilli, later identified as Haemophilus influenzae with the patient commenced on ceftriaxone. The isolate was beta-lactamase negative and antibiotic susceptibility testing (AST) using disc diffusion revealed the isolate resistant to ceftriaxone and ampicillin by EUCAST methodology, with the patient subsequently changed to amoxicillin/clavulanate. Further AST using the CLSI methodology in parallel demonstrated discrepant results between the two susceptibility methods. The patient recovered without complications. Conclusion: This discrepancy could lead to inconsistent reporting of susceptibilities between laboratories, and consequently antibiotic prescribing, especially for invasive isolates. As more laboratories adopt EUCAST methodologies for AST interpretation in Australia and globally, it is important for clinicians to consider the clinical implications of these methodological discrepancies.

Synergistic activity of Thymus capitatus essential oil and cefotaxime against ESBL-producing Klebsiella pneumoniae.

The objective of the current study was to evaluate the interaction between Tunisian Thymus capitatus essential oil (EO) and cefotaxime against Extended-Spectrum Beta-lactamases (ESBLs) producing Klebsiella pneumoniae hospital strains. GC-MS revealed that the major component of EO was found to be carvacrol (69.28%). The EO exerts an advanced bactericidal effect against all strains. Synergy between EO and cefotaxime was obtained by combined disk diffusion and checkerboard techniques. Combined use of EO and cefotaxime reduced the MIC of imipenem by 8- to 128-fold for all strains (fractional inhibitory concentration index ˂ 0.5, synergy). The time kill curve assay confirmed the advanced activity of combinatory effects of EO and cefotaxime, with total reduce of bacterial number (CFU/mL) after 6 h of culture. Synergistic activity of the combination between EO and cefotaxime constitute an important strategy as therapeutical option to combat infections caused by ESBLs producing Klebsiella pneumoniae.

Antibacterial activity of Lagerstreomia speciosa and its active compound, corosolic acid, enhances cefotaxime inhibitory activity against Staphylococcus aureus.

AIMS: Various epidemiology studies have reported the emergence of Staphylococcus aureus and its methicillin resistance strain causing global health concerns, especially during and post-COVID-19 pandemic. This pathogen presents as a co-infection in patients with COVID-19. In addition, certain virulence factors and resistance to ß-lactam antibiotics, including cefotaxime, have been identified. We aimed to investigate the antibacterial activity of Lagerstreomia speciosa, a medicinal plant with antidiabetic activity, against S. aureus, including the strain resistant to methicillin. Furthermore, we examined whether the extract and one of its bioactive compounds, corosolic acid, can enhance the therapeutic effect of cefotaxime on antibiotic-resistant S. aureus. METHODS AND RESULTS: The minimum inhibitory concentration of each substance was determined using the standard broth microdilution test following the checkerboard dilution. The type of interactions, synergistic, additivity, indifference, or antagonism, were determined using isobolograms analysis and the dose reduction index (DRI). The evaluation of synergy and bactericidal activity of the natural products in combination with cefotaxime was performed using the time-kill kinetic assay. Corosolic acid, L. speciosa leaves extract, and bark extract alone showed antibacterial activity against all tested S. aureus ATCC 33591, S. aureus ATCC 29213, S. aureus ATCC 25923, and clinical isolated S. aureus. Corosolic acid enhanced the antibacterial activity of cefotaxime, showing a synergistic effect and greater DRI of cefotaxime against all tested S. aureus strains. Time-kill kinetic assay showed that corosolic acid has a more profound effect than L. speciosa extracts to potentiate the bactericidal activity of cefotaxime. Whereas L. speciosa leaves and bark extract showed some inhibitory effect on the growth of S. aureus after a single administration. CONCLUSIONS: Lagerstreomia speciosa leaves and bark extract and its active compound, corosolic acid, could be used as a potential anti-Staphylococcus aureus treatment to enhance the therapeutic use of cefotaxime.

Molecular characterization and antibiotic resistance profile of ESBL-producing Escherichia coli isolated from healthy cow raw milk in smallholder dairy farms in Bangladesh.

Background and Aim: The emergence of antimicrobial-resistant bacteria, such as Escherichia coli in milk, is a serious public health concern as milk is considered a complete food and an important part of daily human diet worldwide, including in Bangladesh. However, there have been no reports on the molecular characterization and antibiotic resistance profile of extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk of healthy cows in Bangladesh. Therefore, this study aimed to detect and characterize ESBL-producing E. coli (ESBL-Ec) in milk samples from healthy cows in smallholder dairy farms in Mymensingh district, Bangladesh, and assess the potential risk of consuming this milk. Materials and Methods: A total of 100 milk samples were collected from apparently healthy cows on smallholder dairy farms. Escherichia coli was isolated from the collected samples using standard methods. The detection of ESBL-Ec was performed phenotypically using cultural methods and genotypically by ESBL genetic determinants using multiplex polymerase chain reaction. Antimicrobial susceptibility testing of the ESBL-Ec isolates was performed using the disk diffusion method with 15 common antimicrobials. Results: In this study, out of the 100 samples tested, 70 (70%) were found to be positive for E. coli. Among these, 41 (58.6%) strains were identified as ESBL-producing, both phenotypically and genotypically, with the presence of blaCTX-M, blaTEM, and blaSHV individually or combined (blaCTX-M plus blaTEM plus blaSHV). The antibiogram of these ESBL-positive isolates revealed high resistance against commonly used antibiotics, such as ampicillin, cefotaxime, and gentamicin (100%), azithromycin (88%), oxytetracycline (27%), nalidixic acid, cotrimoxazole/trimethoprim (24%), and streptomycin (22%). In addition, one isolate showed resistance to 4th generation of cephalosporin (cefepime). Most importantly, extensive multidrug resistance was found in many ESBL-Ec isolates. However, the isolates were highly sensitive to drugs such as ceftriaxone (100%) and imipenem (100%). This is the first study to detect ESBL-Ec in raw milk from healthy cows on smallholder dairy farms in Bangladesh. Conclusion: More than 58% of the E. coli isolated from raw milk of healthy cows tested positive for ESBL production and showed resistance to most commonly used antimicrobials which may be alarming for human health. A limitation of our study is that we had a small size of sample collected from one district in Bangladesh. Therefore, a larger sample size covering a wider geographic area, and using multi-locus sequence typing and whole genome sequencing could provide a more comprehensive understanding of the prevalence and characteristics of ESBL-Ec in Bangladesh.

Prevalence and molecular characterization of cefotaxime-resistant Salmonella strains recovered from retail meat samples in Shenzhen, China, during 2014-2017.

In this work, we collected foodborne Salmonella strains in Shenzhen, China, during 2014-2017 and investigated the genetic profile of all cefotaxime-resistant isolates in the collection. The strains were subjected to antimicrobial susceptibility tests, whole-genome sequencing, bioinformatics analysis, and conjugation studies. A total of 79 cefotaxime-resistant Salmonella were identified and found to exhibit multidrug resistance. Resistance rate recorded during the study period increased from 1.9% to 9.1%. Salmonella Typhimurium was the predominant serovar, and CTX-M family genes were dominant among the ESBLs genes detected. Notably, CTX-M-bearing plasmids or transposons often contain other drug resistance genes. Furthermore, a combination of CTX-M-55 and CTX-M-65 genes was detected for the first time in foodborne Salmonella strains. Our findings reveal the prevalence and molecular characteristics of cefotaxime-resistant foodborne Salmonella strains in southern China. IMPORTANCE Cefotaxime-resistant Salmonella strains pose an increasing threat to human health by causing infections with limited treatment options. It is therefore necessary to undertake a surveillance on the prevalence of such strains and investigate the resistance and transmission mechanisms. In this work, various ESBL genes flanked by different IS located in different mobile genetic elements were detectable among cefotaxime-resistant Salmonella strains. These data show that the high prevalence and genotypic diversity of cefotaxime-resistant foodborne Salmonella strains in China are possibly attributed to the evolution and transmission of a wide range of multidrug resistance-encoding mobile genetic elements.

Alginate/chitosan hydrogels as perspective transport systems for cefotaxime.

This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer-Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime-chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan-cefotaxime complexation considering the influence of a solvent.

Utility of the MBT STAR-Cepha kit in detecting extended-spectrum ß-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. ß-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Uncovering the Important Genetic Factors for Growth during Cefotaxime-Gentamicin Combination Treatment in blaCTX-M-1 Encoding Escherichia coli.

Due to the rapid spread of CTX-M type ESBLs, the rate of resistance to third-generation cephalosporin has increased among Gram-negative bacteria, especially in Escherichia coli, and there is a need to find ways to re-sensitize ESBL E. coli to cephalosporin treatment. A previous study showed that genes involved in protein synthesis were significantly up-regulated in the presence of subinhibitory concentration of cefotaxime (CTX) in a CTX-M-1-producing E. coli. In this study, the interaction between CTX and gentamicin (GEN), targeting protein synthesis, was evaluated in MG1655/pTF2, and the MIC of CTX was strongly reduced (128-fold) in the presence of this combnation therapy. Since the underlying mechanism behind this synergy is not known, we constructed a saturated transposon mutant library in MG1655/pTF2::blaCTX-M-1 containing 315,925 unique transposon insertions to measure mutant depletion upon exposure to CTX, GEN, and combination treatment of CTX and GEN by Transposon Directed Insertion-site Sequencing (TraDIS). We identified 57 genes that were depleted (log2FC ≤ -2 and with q.value ≤ 0.01) during exposure to CTX, 18 for GEN, and 31 for combination treatment of CTX and GEN. For validation, we deleted eight genes that were either uniquely identified in combination treatment, overlapped with monotherapy of GEN, or were shared between combination treatment and monotherapy with CTX and GEN. Of these genes, we found that the inactivation of dnaK, mnmA, rsgA, and ybeD increased the efficacy of both CTX and GEN treatment, the inactivation of cpxR and yafN increased the efficacy of only CTX, and the inactivation of mnmA, rsgA, and ybeD resulted in increased synergy between CTX and GEN. Thus, the study points to putative targets for helper drugs that can restore susceptibility to these important drugs, and it indicates that genes involved in protein synthesis are essential for the synergy between these two drugs. In summary, the study identified mutants that sensitize ESBL-producing E. coli to CTX and a combination of CTX and GEN, and it increased our understanding of the mechanism behind synergy between ß-lactam and aminoglycoside drugs. This forms a framework for developing new strategies to combat infections caused by resistant bacteria.