In Vitro Activity of Ceftolozane-Tazobactam and Other Antibiotics against Pseudomonas aeruginosa Infection-Isolates from an Academic Medical Center in Thailand.

(1) Background: Resistant Pseudomonas aeruginosa (PA) infections have limited treatment options. Data on the activity of ceftolozane-tazobactam (C-T) against PA in Thailand are limited. Objectives: The objective of this study was to identify the in vitro activity of C-T against general and resistant PA isolates from patients with real clinical infections from the HRH Princess Maha Chakri Sirindhorn Medical Center (MSMC) compared to other antibiotics and to study the resistant molecular patterns of those PA strains which were resistant to C-T. (2) Materials and Methods: This was an in vitro susceptibility study of 100 PA isolates plus an additional seven resistant PA isolates collected from MSMC patients. All PA isolates were tested with susceptibility broth (Sensititre™) and C-T minimal inhibitory concentration (MIC) test strips (Liofilchem, Roseto degli, Abruzzi, Italy). The C-T-resistant PA isolates were analyzed for six ß-lactamase genes (blaCTX-M, blaNDM, blaIMP, blaVIM, blaOXA-23 and blaOXA-48) and the mcr-1 gene. (3) Results: A total of 100 PA isolates were collected between January 2020 and January 2021 and between February 2021 and September 2021 for the additional 7 resistant isolates. There were 18 resistant PA isolates (6 MDR, 11 XDR and 1 pan-drug resistant isolate). The overall susceptibility of the initial 100 PA isolates and the 18 resistant PA isolates was 94% and 44.5%, respectively, for C-T. The C-T susceptibility rates for isolates non-susceptible to ceftazidime, piperacillin-tazobactam, carbapenems and antipseudomonal ß-lactams were 65.5%, 69.7%, 50% and 44.5%, respectively. Among the 10 isolates which were resistant to C-T, there were only 3 isolates found to have the resistant gene, which included 1 for blaIMP, 1 for blaVIM and 1 for blaNDM. (4) Conclusions: Although C-T was the best susceptibility antibiotic overall for PA isolates and MDR PA isolates at the MSMC, most of the XDR PA isolates and the PDR PA isolate were not susceptible to C-T. The mechanisms for C-T resistance involved multiple factors including the presence of blaIMP, blaVIM and blaNDM.

Colorimetric aptasensor for detecting Salmonella spp., Listeria monocytogenes, and Escherichia coli in meat samples.

In this study, we successfully developed a simple and rapid method for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli using gold nanoparticles and the aptamer aptasensor. We screened 25 specific DNA aptamer candidates against these pathogens using whole-cell Systematic Evolution of Ligands by EXponential enrichment. Among them, Ap6 was selected due to its low energy minimization values of -12.25 and -27.67 kcal/mol derived from MFold and RNAFold analysis, respectively. The assay presented in this study allowed the visual colorimetric detection of labeled colloidal gold nanoparticles as well as determination of UV absorbance at 625 and 525 nm under optimized conditions. The detection limit of this aptasensor was as less as 105 CFU/ml. A random investigation of 50 meat samples, including ham and chicken sausages, collected from the local market revealed 96% accuracy, 96% specificity, and 100% sensitivity of the assay. The colorimetric aptasensor can accomplish one-step detection without pre-culture, DNA extraction, and amplification. Hence, it is an easy, rapid, specific, and qualitative assay that can be used as a point-of-care testing to directly detect multiplex foodborne pathogens.

Development of a duplex lateral flow dipstick test for the detection and differentiation of Listeria spp. and Listeria monocytogenes in meat products based on loop-mediated isothermal amplification.

Listeria spp. are a group of gram-positive bacteria consisting of 20 species. Among them, Listeria monocytogenes is one of the major species that infects humans since it contaminates raw fruits, vegetables, and many others food products. The conventional methods for the detection of Listeria spp. and L. monocytogenes are time-consuming, taking 5-7 days. Herein, a duplex lateral flow dipstick (DLFD) test combined with loop-mediated isothermal amplification (LAMP) was developed for the identification of Listeria spp. and L. monocytogenes within approximately 45 min with the optimized LAMP reaction times at 63 °C. Under the optimized conditions, the method detection limits (MDL) with reference to genomic DNA and pure culture were 900 femtograms (fg) and 20 cfu/mL, respectively. The LAMP-DLFD showed no cross-reactivity with eighteen - other pathogenic bacteria such as Salmonella spp., Staphylococcus aureus, Escherichia coli, Campylobacter coli, C. jejuni, Enterococcus faecalis, Vibrio cholerae, V. parahaemolyticus, Pseudomonas aeruginosa, Shigella dysenteriae, S. flexneri, Bacillus cereus, Lactobacillus acidophilus, L. casei and Pediococcus pentosaceus. Among 100 samples of food products, LAMP-DLFD demonstrated 100% accuracy when compared to other standard detection methods, such as ISO11290-1, enzyme-linked fluorescent assay (ELFA) technology (VIDAS) and PCR. In conclusion, LAMP-DLFD proved to be highly specific and sensitive assays for screening detection of Listeria spp. and L. monocytogenes.

Rapid Colorimetric Assay for Detection of Listeria monocytogenes in Food Samples Using LAMP Formation of DNA Concatemers and Gold Nanoparticle-DNA Probe Complex.

Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity, and accuracy were 100, 90.20, and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

Development and evaluation of a loop-mediated isothermal amplification combined with au-nanoprobe assay for rapid detection of Mycobacterium tuberculosis.

Loop-mediated isothermal amplification (LAMP) has been proposed as an inexpensive and easy to perform assay for molecular diagnostics. We present a novel strategy for the detection of LAMP amplicons derived from Mycobacterium tuberculosis by the use of Au-nanoprobes. When applied to a total of 93 clinical specimens, the LAMP assay demonstrated sensitivity and specificity higher than that of polymerase chain reaction and culture. The Au-nanoprobe augmented LAMP test platform with its advantages of robust reagents and a simple colorimetric detection method can be adapted easily for the rapid detection of other infectious disease agents at a low cost.

Outbreak of Occupational Brucellosis in a Laboratory Technician at Her Royal Highness Princess Sirindhorn Medical Center, Srinakharinwirot University, Thailand.

Objective: To document laboratory transmission of brucellosis and identify the likely mechanism of transmission of brucellosis at Her Royal Highness (HRH) Princess Sirindhorn Medical Center, Thailand. Material and Method: Using small subunit ribosomal RNA (rRNA) sequencing technique to analyze Brucella melitensis cultured from the first 2 patients of the hospital and an infected laboratory technician, and using brucellosis serologic test to rule out infections in all other involved technicians. Results: We had encountered the first 2 cases of brucellosis. Both had infected from community exposure with goat. The first case had pancreatic abscess and spinal bone involvement with a positive blood culture. The second case presented with fever of unknown origin and had a positive blood culture. A few weeks later, 1 of our laboratory technicians presented with fever, myalgia and fatigue. Blood culture grew B. melitensis. He never had any associated community-acquired risk factors for brucellosis. The presumed mechanism of transmission was an inhalation while taking photographs of the bacterial plate of the first patient. B. melitensis identified from our laboratory technician and both patients were analyzed based on 16S-23S rRNA intergenic transcribed spacer (ITS) region. Results of 16S-23S rRNA ITS sequence testing confirmed a match from all patients and laboratory technician's isolate. All other 10 potentially exposed laboratory technicians were asymptomatic. A brucellosis serologic test was negative in all non-infected technicians but was only positive in the 1 infected technician. Conclusion: This is the first report in Thailand of occupational brucellosis transmitted in microbiologic laboratory. The most likely mechanism is air-borne inhalation of bacterial organisms on culture media in the absence of adequate precautions. Laboratory technicians should handle Brucella cultivation with caution utilizing appropriate measures to prevent inhalation.

Detection of Mycobacterium tuberculosis by using loop-mediated isothermal amplification combined with a lateral flow dipstick in clinical samples.

Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92   % and the specificity was 100   % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

Comparative in vitro activity of sitafloxacin against bacteria isolated from Thai patients with urinary tract infections and lower respiratory tract infections.

OBJECTIVE: To determine comparative in vitro activity of sitafloxacin against clinical isolates of bacteria from Thai patients with urinary tract infection and those with lower respiratory tract infection. MATERIAL AND METHOD: 1,255 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus spp, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis isolated from different Thai patients with urinary tract infection and those with lower respiratory tract infection in 2010 were included. The minimum inhibitory concentrations (MICs) of sitafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, imipenem, amikacin, ampicillin, ceftazidime, ceftriaxone, penicillin, piperacillin/tazobactam, vancomycin, azithromycin and trimethoprim/sulfamethoxazole were determined by standard agar dilution method. RESULTS: The MIC50 and MIC90 values of sitafloxacin against all tested bacteria were lowest when compared with those of levofloxacin, ciprofloxacin and moxifloxacin. Sitafloxacin was active against 51% of methicillin-resistant S. aureus (MRSA) isolates. The activity of sitafloxacin against multidrug-resistant (MDR) Gram-negative bacteria, such as, extended spectrum beta-lactamase (ESBL)-producing E. coli and K. pneumomiae, P. aeruginosa and A. baumannii was comparable to or more than that of some beta-lactam/beta-lactamase inhibitors, cephalosporins or carbapenems. CONCLUSION: Sitafloxacin is more active than levofloxacin, ciprofloxacin and moxifloxacin against isolated bacteria from Thai patients with urinary tract and lower respiratory infections including antibiotic resistant bacteria, such as MRSA, ESBL-producing Gram-negatives, carbapenem-resistant A. baumannii.

Characterization of thermophilic halotolerant Aeribacillus pallidus TD1 from Tao Dam Hot Spring, Thailand.

The bacterial strain TD1 was isolated from Tao Dam hot spring in Thailand. Strain TD1 was Gram positive, rod-shaped, aerobic, motile, and endospore forming. The cell was 2.0-40 µm in length and about 0.4 µm in diameter. The optimum growth occurred at 55-60 °C and at pH 7-8. Strain TD1 was able to grow on medium containing up to 10% NaCl. The DNA G+C content was 38.9 mol%. The cellular fatty acid content was mainly C(16:0), which comprised 25.04% of the total amount of cellular fatty acid. 16S rDNA showed 99% identity to Aeribacillus pallidus DSM 3670(T). Bayesian tree analysis strongly supported the idea that strain TD1 is affiliated with genus Aeribacillus, as Aeribacillus pallidus strain TD1. Although the 16S rDNA of A. pallidus strain TD1 is similar to that of A. pallidus DSM 3670(T), some physiological properties and the cellular fatty acid profiles differ significantly. A. pallidus strain TD1 can produce extracellular pectate lyase, which has not been reported elsewhere for other bacterial strains in the genus Aeribacillus. A. pallidus strain TD1 may be a good candidate as a pectate lyase producer, which may have useful industrial applications.

Purification and characterization of organic solvent and detergent tolerant lipase from thermotolerant Bacillus sp. RN2.

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9-11 and temperature range of 45-60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

Detection of non-amplified Mycobacterium tuberculosis genomic DNA using piezoelectric DNA-based biosensors.

Piezoelectric DNA-based biosensor technology was developed as a new method for detection of M. tuberculosis. This method consists of immobilizing a thiol-modified oligonucleotide probe on the gold electrode surface of a quartz crystal, using a self-assembled monolayer method. The advantage of this study is that a non-amplified genomic bacterial DNA target was used. Instead, the genomic DNA was digested by restriction enzyme to obtain DNA fragments containing the target sequence. The fabricated biosensor was evaluated through an examination of 200 samples. No cross hybridization were observed against M. avium complex and other microorganisms. This target DNA preparation, without PCR amplification, will reduce time, costs, and the tedious step of amplification.

Low cost combination of DCIP and MCV was better than that of DCIP and OF in the screening for hemoglobin E.

OBJECTIVE: To evaluate hemoglobin E screening tests in a large scale of cases. MATERIAL AND METHOD: A cross-sectional descriptive study was conducted Whole blood obtained from subjects was evaluated for CBC, OF, DCIP, and hemoglobin typing. RESULTS: Five hundred twenty seven hemoglobin E and 280 reference subjects participated. DCIP's sensitivity, specificity, positive predictive value, and negative predictive value were 97.16%, 98.93%, 99.42%, and 95.19%, respectively. These values of OF were 69.12%, 80.00%, 86.67%, and 57.88%, respectively. In the combination of DCIP and OF gave rise to these values of 99.43%, 79.29%, 90.03%, and 96.67%, respectively. Finally the combination of DCIP and MCV < 80 fL resulted in these values to be 99.43%, 98.93%, 99.43%, and 98.93%, respectively. False positive and false negative rate were 1.07% and 0.57%, respectively. CONCLUSION: Combination of DCIP and MCVwas better than that of DCIP and OF in hemoglobin E screening.

Cytokine production in NK and NKT cells from Mycobacterium tuberculosis infected patients.

Tuberculosis, a major health problem in developing countries, has re-emerged in recent years in many countries. While it is accepted that various lymphocyte subsets are important responses to mycobacterial infection, the roles of NK and NKT cells in producing cytokines are still unclear. Thus we have evaluated, in Mycobacterium tuberculosis infection, the frequency of cytokine producing cells by flow cytometry. Of 30 individuals examined, 17 had clinical evidence of pulmonary tuberculosis while the rest showed no evidence of infection. Patients had a significantly higher number of IFN-gamma and IL-4-producing T cells compared to control subjects, but the ratio of IFN-gamma to IL-4-producing T cells was similar in both groups. There were no differences between cytokine profiles of NK cells in patients and control subjects. A significant increase in the number of NKT cells was observed in patients. A striking finding was the higher frequency of IL-4-producing NKT cells compared to IFN-gamma-producing cells. Moreover, individual NKT cell produced both IFN-gamma and IL-4. The preferential type of Thl or Th2 cells is due to mycobacterial strain, type of antigen presenting cells and stage of disease, all of which can lead to different patterns of cytokine production by variety of lymphocyte subsets.

Acridine orange staining and viability of the coccoid form of Campylobacter upsaliensis.

Conversion of Campylobacter upsaliensis to the coccoid form during aerobic incubation at 37 degrees C was not prevented by treatment with chloramphenicol and was accompanied by severe decreases in isocitrate dehydrogenase activity and oxygen uptake. Although the coccoid forms fluoresced orange-red by acridine orange staining, agarose gel electrophoresis indicated an extensive degradation of the ribosomal RNA. This suggests that acridine orange staining may not be a good indicator of viability and that the coccoid form of C. upsaliensis at 37 degrees C is degenerative rather than part of the life cycle.

Modified GC medium for identification of group B streptococci.

The modified GC medium (MGC) was developed for identification of beta-hemolytic group B streptococci. This medium was developed on the basis of enhancing-pigment production of group B streptococci. Three hundred and thirty isolates were tested including 180 isolates of beta-hemolytic group B streptococci, 102 isolates of beta-hemolytic non-group B streptococci, and 48 isolates of Enterococcus faecalis. All isolates of group B streptococci gave carotenoid pigment by this medium. On the other hand, all of non-group B streptococci and E. faecalis did not show pigment after 72 h incubation. The specificity and sensitivity of MGC was 100%. There were no false positive and false negative in this medium. The MGC may be the alternative of choice for the presumptive identification of group B streptococci.