Drug Sensitivity Testing Research Articles

FOXM1 is critical for the fitness recovery of chromosomally unstable cells

Tumor progression and evolution are frequently associated with chromosomal instability (CIN). Tumor cells often express high levels of the mitotic checkpoint protein MAD2, leading to mitotic arrest and cell death. However, some tumor cells are capable of exiting mitosis and consequently increasing CIN. How cells escape the mitotic arrest induced by MAD2 and proliferate with CIN is not well understood. Here, we explored loss-of-function screens and drug sensitivity tests associated with MAD2 levels in aneuploid cells and identified that aneuploid cells with high MAD2 levels are more sensitive to FOXM1 depletion. Inhibition of FOXM1 promotes MAD2-mediated mitotic arrest and exacerbates CIN. Conversely, elevating FOXM1 expression in MAD2-overexpressing human cell lines reverts prolonged mitosis and rescues mitotic errors, cell death and proliferative disadvantages. Mechanistically, we found that FOXM1 facilitates mitotic exit by inhibiting the spindle assembly checkpoint (SAC) and the expression of Cyclin B. Notably, we observed that FOXM1 is upregulated upon aneuploid induction in cells with dysfunctional SAC and error-prone mitosis, and these cells are sensitive to FOXM1 knockdown, indicating a novel vulnerability of aneuploid cells.

Study on a galactose decomposition strain of Lactiplantibacillus plantarum MWLp-4 from human milk: Safety evaluating and key genes analysis of potential probiotic function

Galactose intolerance, associated with bloating and abdominal pain, is similar to lactose intolerance. Congenital galactose malabsorption in a child can lead to severe diarrhea. To avoid the consequences of galactose malabsorption, we obtained a galactose decomposition strain of Lactiplantibacillus plantarum MWLp-4 from human milk. Importantly, this strain had almost overlapping dynamics of galactose and lactose utilization, showing a great galactose catabolic ability. In this paper, we studied safety evaluation including hemolysis test, drug sensitivity test, and determination of amine in vitro of L. plantarum MWLp-4. In addition, we evaluated the probiotic function such as antimicrobial properties, adhesion, acid resistance, and bile salt resistance, etc. Meanwhile, we have analyzed the key genes on function and safety. As a conclusion, this strain possess great safety and probiotic function. The genes related to galactose metabolism, bacteriocin synthesis, surface protein synthesis, bile salt hydrolysis and stress response are found in L. plantarum MWLp-4. Analysis of the consistency of the phenotype with its genotype via whole genome sequencing showed that the phenotype of its physiological function is due to the large number of related genes existing in it. Therefore, this study would provide potential degrading galactose probiotics for producing special functional food.

Real-time monitoring ATP variation in human cancer organoids for a long term by DNA-based nanosensor

Cancer organoids have become promising tools for predicting drug responses on many different types of cancer. Detecting the adenosine triphosphate (ATP) has currently been considered as a decisive test to profile the growth status and drug responses of organoids. ATP profiling using commercial ATP detection kits, which involve cell lysis, can be performed at a single time spot, causing a clinical dilemma of selecting the optimal time spot to adopt diverse cancer types and patients. This study provides a feasible solution to this dilemma by developing a DNA-based ATP nanosensor to realize real-time ATP monitoring in organoids for a long term. The employment of DNA materials ensures high biocompatibility and low cytotoxicity, which are crucial for fragile organoids; The usage of tetrahedral DNA framework ensures cell permeability and intracellular ATP detection; The introduction of ATP-mediated molecular replacement ensures the high sensitivity and selectivity of ATP recognition. These features result in the first successful attempt on real-time monitoring ATP in organoids for up to 26 days and gaining growth status curves for the whole duration of a drug sensitivity test on human lung cancer organoids.

Identification of collateral sensitivity and evolutionary landscape of chemotherapy-induced drug resistance using cellular barcoding technology.

Background: One of the most significant challenges impeding cancer treatment effectiveness is drug resistance. Combining evolutionary understanding with drug resistance can pave the way for the identification of second-line drug options that can overcome drug resistance. Although capecitabine and irinotecan are commonly used therapeutic agents in the treatment of CRC patients, resistance to these agents is common. The underlying clonal dynamics of resistance to these agents using high-resolution barcode technology and identification of effective second-line drugs in this context remain unclear. Methods and materials: Caco-2 and HT-29 cell lines were barcoded, and then capecitabine and irinotecan resistant derivatives of these cell lines were established. The frequencies of barcodes from resistant cell lines and harvested medium, longitudinally, were determined. Collateral drug sensitivity testing was carried out on resistant Caco-2 and HT-29 cell lines using single agents or drug combinations. The SyngeryFinder tool was used to analyse drug combination testing. Results: In Caco-2 and HT-29 cell lines, barcode frequency measurements revealed clonal dynamics of capecitabine and irinotecan formed by both pre-existing and de novo barcodes, indicating the presence of polyclonal drug resistance. The temporal dynamics of clonal evolution in Caco-2 and HT-29 cell lines were demonstrated by longitudinal analysis of pre-existing and de novo barcodes from harvested medium. In Caco-2 and HT-29 cell lines, collateral drug sensitivity revealed a number of drugs that were effective alone and in combination. Conclusion: The use of barcoding technology reveals the clonal dynamics of chemotherapy-induced drug resistance not only from harvested cell populations, but also from longitudinal sampling throughout the course of clonal evolution. Second-line drugs that sensitize drug-resistant CRC cell lines are identified through collateral drug testing.

New insights into the antimicrobial action and protective therapeutic effect of tirapazamine towards Escherichia coli-infected mice

Escherichia coli is an important pathogen responsible for numerous cases of diarrhoea worldwide. The bioreductive agent tirapazamine (TPZ), which was clinically used to treat various types of cancers, has obvious antibacterial activity against E. coli strains. In the present study, we aimed to evaluate the protective therapeutic effects of TPZ in E. coli-infected mice and provide insights into its antimicrobial action mechanism. The MIC and MBC tests, drug sensitivity test, crystal violet assay and proteomic analysis were used to detect the in vitro antibacterial activity of TPZ. The clinical symptoms of infected mice, tissue bacteria load, histopathological features and gut microbiota changes were regarded as indicators to evaluation the efficacy of TPZ in vivo. Interestingly, TPZ-induced the reversal of drug resistance in E. coli by regulating the expression of resistance-related genes, which may have an auxiliary role in the clinical treatment of drug-resistant bacterial infections. More importantly, the proteomics analysis showed that TPZ upregulated 53 proteins and downregulated 47 proteins in E. coli. Among these, the bacterial defence response-related proteins colicin M and colicin B, SOS response-related proteins RecA, UvrABC system protein A, and Holliday junction ATP-dependent DNA helicase RuvB were all significantly upregulated. The quorum sensing-related protein glutamate decarboxylase, ABC transporter-related protein glycerol-3-phosphate transporter polar-binding protein, and ABC transporter polar-binding protein YtfQ were significantly downregulated. The oxidoreductase activity-related proteins pyridine nucleotide-disulfide oxidoreductase, glutaredoxin 2 (Grx2), NAD(+)-dependent aldehyde reductase, and acetaldehyde dehydrogenase, which participate in the elimination of harmful oxygen free radicals in the oxidation-reduction process pathway, were also significantly downregulated. Moreover, TPZ improved the survival rate of infected mice; significantly reduced the bacteria load in the liver, spleen, and colon; and alleviated E. coli-associated pathological damages. The gut microbiota also changed in TPZ-treated mice, and these genera were considerably differentiated: Candidatus Arthromitus, Eubacterium coprostanoligenes group, Prevotellaceae UCG-001, Actinospica, and Bifidobacterium. TPZ may represent an effective and promising lead molecule for the development of antimicrobial agents for the treatment of E. coli infections.

Evaluation of pathogen distribution and antimicrobial resistance in breast plastic surgery infection.

The demand for mammaplasty has increased in recent years, and infection remains one of the common and serious post-operative complications. In this study, we analyzed the pathogen distribution and antibiotic susceptibility of breast plastic surgery infections, and compared the differences in pathogenic species between surgical procedures. The number of each species was counted in the microbial samples of breast plastic surgery infections in Plastic Surgery Hospital of Chinese Academy of Medical Sciences from January 2011 to December 2021. The in vitro antibiotic sensitivity testing data were analyzed using WHONET 5.6 software. The surgical techniques, the period of infection, and other details were gathered in accordance with the clinical data. There were a total of 42 cases included, and 43 different types of pathogenic bacteria, mostly gram-positive bacteria, were found. CoNS (13/43) and Staphylococcus aureus (22/43) made up the majority. The most prevalent of the five Gram-negative bacteria was Pseudomonas aeruginosa. Results of drug sensitivity tests indicate that S. aureus is highly sensitive to vancomycin, cotrimoxazole, and linezolid, whereas CoNS is highly sensitive to vancomycin, linezolid, and chloramphenicol. Both of these bacteria show high resistance to erythromycin and penicillin. Breast augmentation, breast reconstruction, and breast reduction surgery were the most frequently associated breast surgery procedures in this study with infections, with the highest number of infections occurring following breast augmentation with fat grafting, breast reduction surgery, and breast reconstruction with autologous tissue. Various breast plastic surgery procedures have different common pathogens of infection, but the most prevalent are CoNS and S. aureus. Additionally, the majority of the infections in this study were in the early stages. Gram-positive bacteria were the predominant cause of breast plastic surgery infections, and the types of infection strains, the period of infection onset, and the antibiotic susceptibility of prevalent strains varied between breast plastic procedures.

ZNF765 is a prognostic biomarker of hepatocellular carcinoma associated with cell cycle, immune infiltration, m6A modification, and drug susceptibility.

Hepatocellular carcinoma (HCC) is an ongoing challenge worldwide. Zinc finger protein 765 (ZNF765) is an important zinc finger protein that is related to the permeability of the blood-tumor barrier. However, the role of ZNF765 in HCC is unclear. This study evaluated the expression of ZNF765 in hepatocellular carcinoma and the impact of its expression on patient prognosis based on The Cancer Genome Atlas (TCGA). Immunohistochemical assays (IHC) were used to examine protein expression. Besides, a colony formation assay was used to examine cell viability. We also explored the relationship between ZNF765 and chemokines in the HCCLM3 cells by qRT-PCR. Moreover, we examined the effect of ZNF765 on cell resistance by measurement of the maximum half-inhibitory concentration. Our research revealed that ZNF765 expression in HCC samples was higher than that in normal samples, whose upregulation was not conducive to the prognosis. The results of GO, KEGG, and GSEA showed that ZNF765 was associated with the cell cycle and immune infiltration. Furthermore, we confirmed that the expression of ZNF765 had a strong connection with the infiltration level of various immune cells, such as B cells, CD4+ T cells, macrophages, and neutrophils. In addition, we found that ZNF765 was associated with m6A modification, which may affect the progression of HCC. Finally, drug sensitivity testing found that patients with HCC were sensitive to 20 drugs when they expressed high levels of ZNF765. In conclusion, ZNF765 may be a prognostic biomarker related to cell cycle, immune infiltration, m6A modification, and drug sensitivity for hepatocellular carcinoma.

Strain Identification and Drug Resistance Analysis of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry in Nontuberculous Mycobacterial Lung Disease.

To evaluate the clinical value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in detecting Nontuberculous mycobacteria (NTM). The clinical data of 172 patients with suspected NTM lung disease were collected from our hospital from January 1, 2018, to December 30, 2021. The results were compared with those of BACTEC MGIT 960 in liquid culture and gene chip. This study also utilised MALDI-TOF MS to detect macrolide (MA) and amikacin (Am) mutations. One hundred thirty-seven cases of NTM pulmonary disease were confirmed by identifying the NTM gene chip in bronchoalveolar lavage fluid and/or MALDI-TOF MS detection. The positive predictive value and negative predictive value were 100% (131/131) and 85.37% (35/41), respectively, and the consistency of the two methods was high (kappa=0.899). For the drug resistance detection of MAs, the consistency rate between MALDI-TOF MS detection and drug sensitivity detection was 97.71% (128/131), the sensitivity was 81.25% (13/16) and the specificity was 100% (115/115). The positive and negative predictive values were 100% (13/13) and 93.75% (115/118), respectively. There was no coincidental consistency between the two methods, and the consistency was high (P<0.001, kappa=0.884). For the drug resistance test of Am, the consistency rate between the MALDI-TOF MS test and the drug sensitivity test was 93.13% (122/131), the sensitivity was 93.52% (101/108), the specificity was 90.91% (21/23) and the positive predictive value and negative predictive value were 98.06% (101/103) and 75.00% (21/28), respectively. The two methods had high consistency, and the consistency was not coincidental (P<0.001, kappa=0.781). Utilising MALDI-TOF MS has a good consistency with the drug resistance gene chip method and can be a rapid and effective method to identify strains and drug resistance of NTM. Therefore, it has certain clinical application value in patients with suspected NTM lung disease.

Antimicrobial Effects of Sophora flavescens Alkaloids on Metronidazole-Resistant Gardnerella vaginalis in Planktonic and Biofilm Conditions

Bacterial vaginosis (BV) is a common infectious disease of the lower female reproductive tract, which is characterized by the augmentation of anaerobic bacteria. Gardnerella (G.) vaginalis plays a predominant role in BV recurrence relating to its higher virulence potential and biofilm formation ability. With the increased proportion of metronidazole-resistant G. vaginalis, controlling resistance to metronidazole and finding more effective drugs became a major concern. In this study, 30 clinical strains were cultured from the vaginal secretions of BV patients, followed by PCR and 16S rDNA sequencing identification. According to the CLSI guidelines for anaerobic drug sensitivity testing, 19 strains were identified as metronidazole-resistant (minimum inhibitory concentration, MIC ≥ 32 μg/mL), of which 4 clinical strains were observed to be strong biofilm producer and the final minimum biofilm inhibitory concentration (MBIC) of metronidazole was increased to 512 μg/mL. Sophora flavescens Alkaloids (SFAs), a traditional chinese medicine, could not only inhibit the growth of metronidazole-resistant G. vaginalis in planktonic (MIC: 0.3125–1.25 mg/mL), but also eliminate the biofilm formation (MBIC: 0.625–1.25 mg/mL). In the high-magnification scanning electron, it was observed that the morphology of biofilm changed from a thick to flaky shape and was nearly depleted. These results indicate that SFAs could not only inhibit the growth of metronidazole-resistant G. vaginalisin planktonic and biofilm levels, but also destroyed the biofilm morphology and microstructure, which may contribute to the prevention of BV recurrence.

Microbial distributions in the conjunctiva and lacrimal sacs of patients with chronic dacryocystitis in a tertiary hospital.

The aim was to clarify the distributions of bacteria in the conjunctival sac and lacrimal sac in patients with chronic dacryocystitis. In total, 297 (322 eyes) chronic dacryocystitis patients who underwent nasal endoscopic dacryocystorhinostomy (EN-DCR) were included. Conjunctival sac secretions from the affected eye were collected preoperatively, and lacrimal sac retention fluid from the affected side in the same patient was collected intraoperatively. Bacterial culture and drug sensitivity testing were performed to determine bacterial distributions. In total, 127 bacterial isolates (49 species) were detected in 123 eyes in the conjunctival group, with a positivity rate of 38.2% (123/322); 85 bacterial isolates (30 species) were detected in 85 eyes in the lacrimal sac group, with a positivity rate of 26.4% (85/322). The positivity rates were significantly different (P = 0.001) between two groups. The gram-negative bacilli proportion in the lacrimal sac group (36/85, 42.4%) was significantly higher than that in the conjunctival sac group (37/127, 29.2%) ( P = 0.047). Positive conjunctival sac secretion culture (123/322) was significantly associated with increased ocular secretion (281/322, 87.3%) (P = 0.002). Among the culture-positive bacteria in the conjunctival sac group and the lacrimal sac group, 30/127, 23.6% and 43/127, 26.7% and 21/85, 24.7% and 20/85, 23.5% were resistant to levofloxacin and tobramycin, respectively. This study illustrated differences in bacterial distributions between conjunctival sac secretions and retained lacrimal sac fluid in chronic dacryocystitis patients, with a higher proportion of gram-negative bacilli in lacrimal sac secretions. The ocular surface flora in chronic dacryocystitis patients is partially resistant to levofloxacin and tobramycin, which need to be considered by ophthalmologists.

Effect of Stenotrophomonas maltophilia on Tuberculosis.

Tuberculosis (TB) is an important infectious disease suffered by many countries, including China. In this stage, accurate diagnosis and treatment are key measures for the prevention and control of TB. Stenotrophomonas maltophilia is a global emerging Gram-negative, multidrug-resistant (MDR) organism characterized by its high contribution to the increase in crude mortality rates. By single cell preparation and strain identification, we isolated S. maltophilia from stored cultures of Mycobacterium tuberculosis (Mtb). We found that S. maltophilia could not be removed from sputum by alkali treatment or inhibited by antibiotic mixture added to MGIT 960 indicator tubes. When co-cultured with Mtb on a Löwenstein-Jensen (L-J) slant, it could inhibit the growth of Mtb and liquefy the medium. More seriously, it was resistant to 10 of the 12 anti-TB drugs, including isoniazid and rifampin, and made the mixed samples display multidrug-resistant Mtb (MDR-TB) results in the drug sensitivity test, which might change a treatment regimen and increase disease burden. Following, we conducted a small-scale surveillance which showed that the isolation rate of S. maltophilia in TB patients was 6.74%, but these patients had no special characteristics and the presence of S. maltophilia was hidden. The effect of S. maltophilus on TB and its mechanism are unclear and require more attention. IMPORTANCE China is a high-burden country for tuberculosis (TB), multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB), and HIV-associated TB. Increasing the positive rate of culture and the accuracy of antibiotic susceptibility testing (AST) are important for diagnosis, treatment, and control of TB. In our study, we found that the isolation rate of Stenotrophomonas maltophilia in TB patients was not neglectable and that this bacterium affects the isolation and AST results of TB. Due to a lack of relevant research, the impact of S. maltophilia on the course and outcome of TB is unclear. However, the characteristics of S. maltophilia that increase disease mortality require attention. Therefore, in the clinical testing of TB, in addition to mycobacteria, it is recommended to increase the detection of co-infected bacteria and improve the awareness of TB clinicians of these bacteria.

Analysis of tuberculosis epidemiological characteristics and drug resistance among the floating population in Beijing in 2019

Objective: To analyze the epidemic characteristics and drug resistance of pulmonary tuberculosis among the floating population in Beijing and to provide a scientific basis for formulating strategies for the prevention and control of tuberculosis among the floating population. Methods: Data of tuberculosis patients who were positive for Mycobacterium tuberculosis culture was collected from 16 districts and one municipal institution of tuberculosis control and prevention in Beijing in 2019. The strain samples were tested for drug sensitivity by the proportional method. According to household registration location, patients were divided into the floating population and Beijing registration. SPSS 19.0 software analyzed tuberculosis patients' epidemic characteristics and drug resistance in the floating population. Results: In 2019, there were 1 171 culture-positive tuberculosis patients in Beijing, among the floating population, 593 (50.64%) patients were identified, with a male-to-female sex ratio of 2.2∶1 (409∶184). Compared to patients under household registration as Beijing residents, a higher proportion of young adults aged 20-39 years (65.09%,386/593) were noticed, with 55.65% (330/593) reported from the urban areas and 96.80% (574/593) were reported the first time. The differences were statistically significant (all P<0.05). After completing the drug sensitivity test, 37 cases were with multiple drug-resistant tuberculosis, accounting for 6.24% (37/593). The rates of isoniazid resistance (42.11%,8/19) and multidrug resistance (21.05%,4/19) in floating population patients after retreatment were significantly higher than those in newly treated patients (11.67%, 67/574 and 5.75%, 33/574), and the differences were statistically significant (all P<0.05). Conclusions: Most patients with tuberculosis in the floating population in Beijing in 2019 were young males aged 20-39 years. The reporting areas were urban areas and the newly treated patients mainly. The patients with tuberculosis in the re-treated floating population were more likely to suffer from multidrug and drug resistance, which should be taken as the key population for prevention and control.

Creation of EGD-Derived Gastric Cancer Organoids to Predict Treatment Responses.

Gastric adenocarcinoma (GAd) is the third leading cause of cancer-related deaths worldwide. Most patients require perioperative chemotherapy, yet methods to accurately predict responses to therapy are lacking. Thus, patients may be unnecessarily exposed to considerable toxicities. Here, we present a novel methodology using patient-derived organoids (PDOs) that rapidly and accurately predicts the chemotherapy efficacy for GAd patients. Methods:Endoscopic GAd biopsies were obtained from 19 patients, shipped overnight, and PDOs were developed within 24 h. Drug sensitivity testing was performed on PDO single-cells with current standard-of-care systemic GAd regimens and cell viability was measured. Whole exome sequencing was used to confirm the consistency of tumor-related gene mutations and copy number alterations between primary tumors, PDOs, and PDO single-cells. Results:Overall, 15 of 19 biopsies (79%) were appropriate for PDO creation and single-cell expansion within 24 h of specimen collection and overnight shipment. With our PDO single-cell technique, PDOs (53%) were successfully developed. Subsequently, two PDO lines were subjected to drug sensitivity testing within 12 days from initial biopsy procurement. Drug sensitivity assays revealed unique treatment response profiles for combination drug regimens in both of the two unique PDOs, which corresponded with the clinical response. Conclusions:The successful creation of PDOs within 24 h of endoscopic biopsy and rapid drug testing within 2 weeks demonstrate the feasibility of our novel approach for future applications in clinical decision making. This proof of concept sets the foundation for future clinical trials using PDOs to predict clinical responses to GAd therapies.

Comparison of two supporting matrices for patient-derived cancer cells in 3D drug sensitivity and resistance testing assay (3D-DSRT).

Central to the success of functional precision medicine of solid tumors is to perform drug testing of patient-derived cancer cells (PDCs) in tumor-mimicking ex vivo conditions. While high throughput (HT) drug screening methods have been well-established for cells cultured in two-dimensional (2D) format, this approach may have limited value in predicting clinical responses. Here, we describe the results of the optimization of drug sensitivity and resistance testing (DSRT) in three-dimensional (3D) growth supporting matrices in a HT mode (3D-DSRT) using the hepatocyte cell line (HepG2) as an example. Supporting matrices included widely used animal-derived Matrigel and cellulose-based hydrogel, GrowDex, which has earlier been shown to support 3D growth of cell lines and stem cells. Further, the sensitivity of ovarian cancer PDCs, from two patients included in the functional precision medicine study, was tested for 52 drugs in 5 different concentrations using 3D-DSRT. Shortly, in the optimized protocol, the PDCs are embedded with matrices and seeded to 384-well plates to allow the formation of the spheroids prior to the addition of drugs in nanoliter volumes with acoustic dispenser. The sensitivity of spheroids to drug treatments is measured with cell viability readout (here, 72 h after addition of drugs). The quality control and data analysis are performed with openly available Breeze software. We show the usability of both matrices in established 3D-DSRT, and report 2D vs 3D growth condition dependent differences in sensitivities of ovarian cancer PDCs to MEK-inhibitors and cytotoxic drugs. This study provides a proof-of-concept for robust and fast screening of drug sensitivities of PDCs in 3D-DSRT, which is important not only for drug discovery but also for personalized ex vivo drug testing in functional precision medicine studies. These findings suggest that comparing results of 2D- and 3D-DSRT is essential for understanding drug mechanisms and for selecting the most effective treatment for the patient.

Pneumatosis in cervical fascial space and necrotizing fasciitis of neck

Backgroud Pneumatosis in the cervical fascial space is a common imaging manifestation of cervical necrotizing fasciitis. At present, although there are some literature reports on pneumatosis in the cervical necrotizing fasciitis, there are few comparative reports. Objective To compare the imaging findings of necrotizing fasciitis of the neck and other cervical space infections, and explore the relationship between pneumatosis in cervical fascial space and necrotizing fasciitis of the neck. Methods A retrospective study was conducted on 56 cases of cervical fascia space infection in our department from May 2015 to March 2021, including 22 cases of necrotizing fasciitis and 34 cases of non-necrotizing fasciitis. 22 cases underwent incision, debridement and catheter drainage in the necrotizing fasciitis group. 26 cases underwent incision, debridement and catheter drainage, and 8 cases underwent ultrasound-guided puncture biopsy and catheter drainage in the non-necrotizing fasciitis group. All cases were confirmed by operation or pathological biopsy and took purulent secretions for bacterial culture and drug sensitivity test during or after operation. All cases were examined by neck CT or MRI before operation. The previous history of surgical incision or puncture and the rupture of cervical space infection were excluded. Results In 22 cases of necrotizing fasciitis, there were 19 cases had air accumulation in the fascial space (86.4%); In 34 cases of non necrotizing fasciitis, 2 cases had air accumulation in the fascial space (5.9%). There was significant difference between the two groups (χ2 = 36.9141, p < .01). Bacterial culture results were positive in 18 (81.8%) patients in the necrotizing fasciitis group. In the non necrotizing fasciitis group, 12 (35.3%) patients had positive bacterial culture results. There was significant difference in the positive rate of bacterial culture between the two groups (χ2 = 11.6239, p < .01). Except one death in necrotizing fasciitis group, all other patients were cured. There was no recurrence after follow-up for 3–6 months. Conclusions and Significance The pneumatosis of necrotizing fasciitis in the neck is dramatically more than that of other infectious diseases. It is suggested that pneumatosis in cervical fascial space is of great significance in the diagnosis of cervical necrosis, the gas production of bacteria may be closely related to the pathogenesis and development of necrotizing fasciitis of the neck, and early measures to block the generation and dissemination of gas is of great significance for treatment.

Clinical Distribution and Drug Resistance of Acinetobacter baumannii in a Hospital from 2019 to 2021

Objective: To analyze the clinical distribution and drug resistance of Acinetobacter baumannii (AB) and provide reference for the treatment of AB infection. Methods: AB isolated from clinical specimens of Huaihua First People’s Hospital from 2019 to 2021 were collected and identified by VITEK 2 Compact, an automated microbial identification and susceptibility testing system, in which drug sensitivity test was also performed. Excel was used for statistical analysis. Results: Among the 1,311 AB strains, 81.16% (1,064 strains) were from sputum samples, and the departments with the highest detections rates of AB were neurosurgery (24.33%), intensive care (15.48%) and infectious disease (11.44%). The drug sensitivity test showed that the resistance rate of 1,311 AB strains to compound sulfamethoxazole and amikacin was 28.38% and 20.54%, respectively, and the resistance rate to 10 other kinds of common antibiotics was more than 40%. Conclusion: The 1,311 AB strains isolated were widely distributed in clinical settings and had strong resistance to commonly used antibiotics. Therefore, it is necessary to strengthen the monitoring of pathogens and drug resistance, formulate reasonable and effective infection control measures, and ensure that antibiotics are used in a reasonable manner.

Isolation of Mucorspp from patients with otitis media and its sensitivity to antifungals

The current study dealt with patients with otitis media, where the fungus was isolated from different pathological conditions, and the patients were distributed according to age, sex, residence, and chronic diseases.The fungal species isolated from these patients were diagnosed, and the focus was on Mucor as it is dangerous and may cause black fungus disease, as it was diagnosed phenotypically and molecularly by polymerase chain reaction (PCR) and a drug sensitivity test was conducted. The results showed that men are more susceptible to infection than women, and adults are more susceptible than adolescents and children, and chronic diseases increased the incidence.The study showed an average ability of Mucor spp to produce hydrolytic enzymes, which makes it dangerous only for patients with low immunity. The study also showed that the fungus is resistant to antifungals except for fluconazole, amphotericin B, and ketoconazole, where the diameter of inhibition was 32, 14, and 10 mm, respectively.

A Rapid Method to Screen for Multi Drug Resistance (MDR) Trait in Clinical Isolates of Acinetobacter baumannii-Acyl Homoserine Lactone (AHL) Screening in CRAB and CSAB Isolates

Aims: Our proposal aimed to evaluate Acyl Homoserine Lactones (AHL) as a functional marker for Multi drug resistant (MDR) potential in clinical isolates of Acinetobacter baumannii. We investigated the AHL production potential of clinical isolates using a biosensor assay directly on a commonly used agar media.&#x0D; Place and Duration of Study: Department of Molecular Diagnostics and Biomarkers, Gleneagles Global Hospitals, Lakdikapul, Hyderabad-500004.&#x0D; Methodology: Antimicrobial drug sensitivity testing (AST) was performed on 72 clinical isolates of A. baumannii against two front-line antibiotics, Imipenem (10µg) and Meropenem (10µg), by Kirby-Bauer disk diffusion method. Production of long chain Acyl Homoserine lactone (AHLs) in the clinical isolates of A. baumannii was tested by cross streaking with the biosensor Chromobacterium violaceum mutant strain CV026 and Agrobacterium tumefaciens (NTL4pZLR4) by agar plate diffusion assay. Screening and identification of the quorum sensing mediator gene abaI was done by PCR to confirm its presence in all the 72 clinical isolates.&#x0D; Results: Out of the 72 clinical isolates, 58 were Carbapenem resistant Acinetobacter baumannii (CRAB) and 14 were Carbapenem sensitive Acinetobacter baumannii (CSAB) for AST by agar disc diffusion method. None of our isolates produced short chain AHLs whereas all the isolates could produce varying amounts of long chain AHLs. Genotypic confirmation of AHL gene was obtained by abaI gene PCR.&#x0D; Conclusion: Carbapenems are the front-line antibiotics used to treat gram negative bacterial infections in emergencies and in the critical care units of hospitals. Clinical isolates A. baumannii has innate resistance to several antibiotics due to various mechanisms, biofilms forming the first line of defense against antibiotics for the bacterium. Our study used AST to carbapenem as the leading marker for MDR, assuming the innate resistance of A. baumannii to other beta lactam antibiotics. Our study brought out certain important observations namely: a) All clinical isolates of A. baumannii produced Quorum Sensing signal molecules, the AHLs b) the clinical isolates of A. baumannii did not produce any short chain AHLs b) All the clinical isolates of A. baumannii produced long chain AHLs c) AHL production is not specific to carbapenem drug resistance because even CSAB isolates produced AHL d) AHL production is inherent to all clinical isolates of A. baumannii and it apparently indicates an underlying biofilm potential and MDR trait in these A. baumannii isolates. e) AHLs could be a universal marker for revealing MDR trait and biofilm potential in clinical microbiology AST profiling protocols.

Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

Significance of the determination of DNA load of drug-resistant mycoplasma pneumoniae and 23sRNA gene mutation locus in children

Purpose: To determine the significance of the detection of Mycoplasma pneumoniae (MP)-DNA load and 23sRNA gene mutation locus in children with drug-resistant MP pneumonia. &#x0D; Methods: A total of 158 children with MP pneumonia received drug sensitivity tests. The patients were divided into resistance group and non-resistance group. The MP-DNA load index (MPLI) and mutation rate of 23sRNA gene at 2063 locus were assessed and compared between the two groups: the MPLI-negative group and the MPLI-positive group, based on whether MPLI was greater than 6.12. The association of MPLI of all the patients and the 23sRNA gene mutation at 2063 locus in the resistance group, as well as clinical indicators were analyzed. &#x0D; Results: The MPLI of the resistance group was lower than that of the non-resistance group. In the MPLI-positive group, the duration of disease, defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity, and length of stay were all longer than those of the MPLI-negative group, while the proportion of cases with extrapulmonary complications and the white blood cell (WBC) count were higher than those of the MPLI-negative group. The mutation rate of 23sRNA gene at 2063 locus in the resistance group was higher than that in non-resistance group (p &lt; 0.05). The defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were longer in the mutation-positive group than those in the mutation-negative group (p &lt; 0.05). &#x0D; Conclusion: The mutation rate of 23sRNA gene at 2063 locus is higher in children with drug-resistant MP pneumonia. Furthermore, low MPLI and 23sRNA gene mutations at 2063 locus are associated with the duration of disease, disappearance time of clinical symptoms and other clinical indicators.