Restoring Ampicillin Sensitivity in Multidrug-Resistant Escherichia coli Following Treatment in Combination with Coffee Pulp Extracts.

Escherichia coli, particularly multidrug-resistant (MDR) strains, is a serious cause of healthcare-associated infections. Development of novel antimicrobial agents or restoration of drug efficiency is required to treat MDR bacteria, and the use of natural products to solve this problem is promising. We investigated the antimicrobial activity of dried green coffee (DGC) beans, coffee pulp (CP), and arabica leaf (AL) crude extracts against 28 isolated MDR E. coli strains and restoration of ampicillin (AMP) efficiency with a combination test. DGC, CP, and AL extracts were effective against all 28 strains, with a minimum inhibitory concentration (MIC) of 12.5-50 mg/ml and minimum bactericidal concentration of 25-100 mg/ml. The CP-AMP combination was more effective than CP or AMP alone, with a fractional inhibitory concentration index value of 0.01. In the combination, the MIC of CP was 0.2 mg/ml (compared to 25 mg/ml of CP alone) and that of AMP was 0.1 mg/ml (compared to 50 mg/ml of AMP alone), or a 125-fold and 500-fold reduction, respectively, against 13-drug resistant MDR E. coli strains. Time-kill kinetics showed that the bactericidal effect of the CP-AMP combination occurred within 3 h through disruption of membrane permeability and biofilm eradication, as verified by scanning electron microscopy. This is the first report indicating that CP-AMP combination therapy could be employed to treat MDR E. coli by repurposing AMP.

Dinactin: A New Antitumor Antibiotic with Cell Cycle Progression and Cancer Stemness Inhibiting Activities in Lung Cancer.

Lung cancer, especially non-small cell lung cancer (NSCLC), is one of the most complex diseases, despite the existence of effective treatments such as chemotherapy and immunotherapy. Since cancer stem cells (CSCs) are responsible for chemo- and radio-resistance, metastasis, and cancer recurrence, finding new therapeutic targets for CSCs is critical. Dinactin is a natural secondary metabolite produced by microorganisms. Recently, dinactin has been revealed as a promising antitumor antibiotic via various mechanisms. However, the evidence relating to cell cycle progression regulation is constrained, and effects on cancer stemness have not been elucidated. Therefore, the aim of this study is to evaluate the new function of dinactin in anti-NSCLC proliferation, focusing on cell cycle progression and cancer stemness properties in Lu99 and A549 cells. Flow cytometry and immunoblotting analyses revealed that 0.1-1 µM of dinactin suppresses cell growth through induction of the G0/G1 phase associated with down-regulation of cyclins A, B, and D3, and cdk2 protein expression. The tumor-sphere forming capacity was used to assess the effect of dinactin on the cancer stemness potential in NSCLC cells. At a concentration of 1 nM, dinactin reduced both the number and size of the tumor-spheres. The quantitative RT-PCR analyses indicated that dinactin suppressed sphere formation by significantly reducing expression of CSC markers (i.e., ALDH1A1, Nanog, Oct4, and Sox2) in Lu99 cells. Consequently, dinactin could be a promising strategy for NSCLC therapy targeting CSCs.

Potential Antimicrobial Properties of Coffee Beans and Coffee By-Products Against Drug-Resistant Vibrio cholerae.

Vibrio cholerae is the causative organism of the cholera epidemic, and it remains a serious global health problem, particularly the multidrug-resistant strain, despite the development of several generic drugs and vaccines over time. Natural products have long been exploited for the treatment of various diseases, and this study aimed to evaluate the in vitro antibacterial activity of coffee beans and coffee by-products against V. cholerae antimicrobial resistant strains. A total of 9 aqueous extracts were investigated, including light coffee (LC), medium coffee (MC), dark coffee (DC), dried green coffee (DGC), dried red coffee (DRC), fresh red coffee (FRC), Arabica leaf (AL), Robusta leaf (RL), and coffee pulp (CP). The influential coffee phytochemicals, i.e., chlorogenic acid (CGA), caffeic acid (CA), and caffeine, were determined using HPLC. The antibacterial properties were tested by agar well-diffusion techniques, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were further determined against 20 V. cholerae isolates. The results revealed that all tested strains were sensitive to coffee extracts, with MIC and MBC values in the range of 3.125-25.0 mg/mL and 12.5-50.0 mg/mL, respectively. With a MIC of 6.25 mg/mL, DGC, DRC, and CP appeared to be the most effective compounds against 65, 60, and 55% of clinical strains, respectively. The checkerboard assay revealed that the combination of coffee extract and tetracycline was greater than either treatment alone, with the fractional inhibitory concentration index (FICI) ranging from 0.005 to 0.258. It is important to note that CP had the lowest FICI (0.005) when combined with tetracycline at 60 ng/mL, which is the most effective dose against V. cholerae six-drug resistance strains (azithromycin, colistin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim), with a MIC of 47.5 µg/mL (MIC alone = 12.5 mg/mL). Time killing kinetics analysis suggested that CA might be the most effective treatment for drug-resistant V. cholerae as it reduced bacterial growth by 3 log10 CFU/mL at a concentration of 8 mg/mL within 1 h, via disrupting membrane permeability, as confirmed by scanning electron microscopy (SEM). This is the first report showing that coffee beans and coffee by-product extracts are an alternative for multidrug-resistant V. cholerae treatment.

Antimicrobial Activity of the Green Tea Polyphenol (-)-Epigallocatechin-3-Gallate (EGCG) against Clinical Isolates of Multidrug-Resistant Vibrio cholerae.

The spread of multidrug-resistant (MDR) Vibrio cholerae necessitates the development of novel prevention and treatment strategies. This study aims to evaluate the in vitro antibacterial activity of green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against MDR V. cholerae. First, MIC and MBC values were evaluated by broth microdilution techniques against 45 V. cholerae strains. The checkerboard assay was then used to determine the synergistic effect of EGCG and tetracycline. The pharmaceutical mode of action of EGCG was clarified by time-killing kinetics and membrane disruption assay. Our results revealed that all of the 45 clinical isolates were susceptible to EGCG, with MIC and MBC values in the range of 62.5-250 µg/mL and 125-500 µg/mL, respectively. Furthermore, the combination of EGCG and tetracycline was greater than either treatment alone, with a fractional inhibitory concentration index (FICI) of 0.009 and 0.018 in the O1 and O139 representative serotypes, respectively. Time-killing kinetics analysis suggested that EGCG had bactericidal activity for MDR V. cholerae after exposure to at least 62.5 µg/mL EGCG within 1 h. The mode of action of EGCG might be associated with membrane disrupting permeability, as confirmed by scanning electron microscopy. This is the first indication that EGCG is a viable anti-MDR V. cholerae treatment.

Spread of Antimicrobial-Resistant Salmonella from Poultry to Humans in Thailand.

Food animal production is important for every country. Several antibiotic agents are used in poultry farming to reduce the economic losses arising from mostly untested infectious diseases. This continued study was performed to determine the prevalence of antibiotic-resistant Salmonella in broiler chickens, poultry farmers, and Salmonella bacteremia patients. A total of 121 Salmonella isolates were collected from the Thai provinces of Khon Kaen (65 isolates), Ratchaburi (43 isolates), and Phayao (13 isolates). Salmonella from chicken showed a high rate of resistance to nalidixic acid and tetracycline. Sixty-four percent of Salmonella isolates carried class 1 integrons (intI1 gene-positive). Among the 121 Salmonella isolates, there were 15 serotypes, with S. Enteritidis being the most common. A clonal relationship between the chicken and human isolates was demonstrated by 3 molecular typing methods: enterobacterial repetitive intergenic consensus polymerase chain reaction; pulsed-field gel electrophoresis; and high-throughput multilocus sequence typing. A spread of the sequence type 11 clone was found between chickens and humans. This study revealed a large-scale Salmonella outbreak in Thailand, a link between resistant bacteria from poultry farms and vertical transmission through the food chain, and horizontal transmission of resistance genes. These results can be used for future surveillance and monitoring.

Prevalence of blaCTX-M and Emergence of blaCTX-M-5-Carrying Escherichia coli in Chrysomya megacephala (Diptera: Calliphoridae), Northern Thailand.

This study was undertaken to assess the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) among blow fly (Chrysomya megacephala) populations in Northern Thailand. Of 600 blow flies collected from rural (n = 400) and urban (n = 200) areas, 334 blow flies carried ESBL-EC (55.7%). Prevalence of ESBL-EC in blow flies captured from rural areas was significantly higher than that from urban region (72.5% vs. 22.0%, p < 0.001). Susceptibility tests revealed that 68.6% of ESBL-EC possessed multidrug-resistant phenotypes. Coresistance to gentamicin (85%) was common, while resistance to ciprofloxacin was relatively low (18.0%). Of the 334 isolates, 253 isolates (75.7%) harbored blaCTX-M, in which blaCTX-M group 1 was predominant (56.5%), followed by blaCTX-M group 9 (39.1%). Interestingly, a single isolate was found to carry blaCTX-M-5, which resided on the IncA/C conjugative plasmid. This is the first report of blaCTX-M-5 from Thailand and its first identification in blow fly. Pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity among ESBL-EC isolates. Nevertheless, identical and closely related PFGE profiles were detected among isolates within the same regions and the regions which are several kilometers apart, suggesting that clonal transmission has occurred. Moreover, epidemiologically related isolates were observed between ESBL-EC from blow flies and human intestinal tract. This study provides evidences that blow flies, C. megacephala, are important reservoirs for ESBL-EC and could potentially act as vectors for the spread of ESBL-EC in a Thai community.

Acquisition of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in intensive care units in Thailand.

This study aimed to assess the prevalence and associated risk factors for extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) acquisition among patients staying in medical and surgical Intensive Care Units (ICU) in Northern Thailand. Rectal swabs were collected from 206 ICU patients upon admission and discharge. Overall, the ESBL-EK acquisition rate among patients during ICU stay was 29.6%. Acquisition rate was significantly higher for K. pneumoniae (20.9%) than that of E. coli (12.1%) (p = 0.024). Multivariate logistic regression analysis identified the use of third generation cephalosporin (p = 0.008) as a risk factor for ESBL-EK acquisition. Sixty-eight ESBL-EK isolates (25 E. coli and 43 K. pneumoniae) were recovered. The majority of ESBL-EK isolates (≥88%) were resistant to ceftazidime, cefepime and aztreonam. Fifty-two acquired ESBL-EK isolates (76.5%) were positive for blaCTX-M and 4 K. pneumoniae isolates simultaneously carried blaNDM-1. Our results reveal that ICU patients could acquire ESBL-EK during hospitalization and the use of third generation cephalosporin should be strictly controlled to prevent the acquisition of ESBL-EK among ICU patients.

Risk Factors for Extended-Spectrum ß-Lactamase-Producing Enterobacteriaceae Carriage in Patients Admitted to Intensive Care Unit in a Tertiary Care Hospital in Thailand.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) are important causes of serious infections in intensive care unit (ICU). This study aimed to investigate the risk factors for intestinal carriage of ESBL-PE among patients admitted to ICU, subsequent ESBL-PE infections, and outcomes of these patients. This study prospectively collected rectal swabs from 215 ICU patients in Northern Thailand and ESBL-PE were isolated. A high prevalence of ESBL-PE carriage (134/215, 62.3%) at ICU admission was observed, with Escherichia coli representing the predominant organism (67.5%) followed by Klebsiella pneumoniae (19.4%). Multivariate logistic regression analysis identified chronic renal disease as the independent risk factor for ESBL-PE carriage (p = 0.009; adjusted odds ratio = 4.369; 95% confidence interval = 1.455-13.119). Among colonized patients, 2.2% (3/134) developed ESBL-PE infections during ICU stay. Phylogenetic analysis of E. coli (n = 108) showed that the predominant group was group A (38.0%), followed by groups B1 (17.6%), D (15.7%), B2 (14.8%), C (7.4%), and F (6.5%). Multilocus sequence typing analysis of the pathogenic groups B2, D, and F revealed 11 different sequence types (STs), with ST131 (n = 13) as the most prevalent, followed by ST648 (n = 5), ST38 (n = 4), ST393 (n = 3), and ST1193 (n = 3). These results are of concern since ESBL-PE may be a prerequisite for endogenous infections and potentially disseminate within the hospital. This is the first study describing ESBL-PE carriage among patients at ICU admission and subsequent ESBL-PE infections in Thailand.

Environmental dissemination of mcr-1 positive Enterobacteriaceae by Chrysomya spp. (common blowfly): An increasing public health risk.

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR.

Risk Factors for Gastrointestinal Colonization and Acquisition of Carbapenem-Resistant Gram-Negative Bacteria among Patients in Intensive Care Units in Thailand.

This study was conducted to investigate the prevalence of and risk factors for colonization and acquisition of carbapenem-resistant (CR) Gram-negative bacteria (GNB) among patients admitted to intensive care units (ICUs) in two tertiary care hospitals in northern Thailand. Screening of rectal swab specimens for CR-GNB was performed on patients at ICU admission and discharge. The phenotypes and genotypes of all isolates were determined. Risk factors were analyzed by logistic regression analysis. The overall carriage rate of CR-GNB at admission was 11.6% (32/275), with the most predominant species carried being Acinetobacter baumannii (n = 15), followed by Klebsiella pneumoniae (n = 9). The risk factor for CR-GNB colonization was hospitalization within the previous 6 months (P = 0.002). During the ICU stay, the rate of CR-GNB acquisition was 25.2% (52/206), with the most predominant species carried being A. baumannii (n = 28) and K. pneumoniae (n = 13). Risk factors associated with CR-GNB acquisition were the use of an enteral feeding tube (P = 0.008) and administration of third-generation cephalosporins (P = 0.032) and carbapenems (P = 0.045). The most common carbapenemase genes in A. baumannii and K. pneumoniae were blaOXA-23/51 and blaNDM, respectively. Patient-to-patient transmission was demonstrated in three cases, resulting in the acquisition of CR A. baumannii (2 cases) and K. pneumoniae (1 case) isolates from other patients who were admitted during the same period of time. This is the first Indochinese study screening patients, examining patients for the carriage of CR-GNB, and further demonstrating the transfer of CR-GNB isolates in ICUs. Our study suggests that effective infection control measures are required to limit the spread of CR-GNB within hospitals.

Occurrence of Extended-Spectrum and AmpC-Type ß-Lactamase Genes in Escherichia coli Isolated from Water Environments in Northern Thailand.

Sixty-eight cefotaxime-resistant Escherichia coli isolates were recovered from different water environments in Northern Thailand. Isolates were mostly resistant to ceftazidime and aztreonam (>90%). The most common extended-spectrum ß-lactamase-encoding gene was blaCTX-M-group 1 (75%) followed by blaCTX-M-group 9 (13.2%). The co-existence of blaCTX-M and AmpC-type ß-lactamase genes was detected in 4 isolates (5.9%). Two E. coli isolates carrying blaCTX-M from canal and river water samples belonged to the phylogenetic group B2-ST131, which is known to be pathogenic. This is the first study on blaCTX-M and blaCMY-2-carrying E. coli and the emergence of ST131 from water environments in Thailand.

Nosocomial spread of class 1 integron-carrying extensively drug-resistant Pseudomonas aeruginosa isolates in a Thai hospital.

Fifty non-duplicate multiresistant isolates of Pseudomonas aeruginosa from a regional hospital in Northern Thailand were investigated for their antimicrobial susceptibility, presence of class 1 integrons and arrangement of gene cassettes as well as their genetic relationships. All but one isolate were classified as extensively drug-resistant P. aeruginosa (XDR-PA). Forty-one isolates (82%) were found to carry class 1 integrons. Amplification of the variable regions of class 1 integrons revealed seven diverse bands ranging in size from 0.7 kb to 7.0 kb. Sequence analysis of class 1 integron variable regions revealed the presence of several gene cassettes associated with resistance to aminoglycosides (aac, aad and aph), including the aac(3)-Ic cassette reported for the first time in Thailand. Gene cassettes encoding resistance to chloramphenicol (cmlA), ß-lactams (bla(PSE), bla(OXA) and bla(VEB)) and rifampicin (arr) were found. The putative small multidrug resistance protein (smr) and an open-reading frame with unknown function (orfD) were also detected. The aadA6-orfD cassette array was the most common integron found in this study. Integron-positive isolates had higher frequencies of antimicrobial resistance than isolates lacking integrons. Pulsed-field gel electrophoresis (PFGE) demonstrated the occurrence of horizontal gene transfer. Interestingly, a large number of XDR-PA isolates carrying identical integrons clearly exhibited the same PFGE pattern, indicating nosocomial spread of these isolates. The presence of XDR-PA carrying class 1 integrons is implicated in the possible spread of drug-resistant organisms, therefore screening for integron-positive P. aeruginosa might be necessary for protection against nosocomial infection.