This work was supported by the Sanming Project of Medicine in Shenzhen (Grant No. SZSM201510050) and the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2022A1515220023). Research Foundation for Advanced Talents of Guandong Provincial People&#39;s Hospital (No. KJ012021097), and the National Natural Science Foundation of China (81871734, 82072380, 82272423). The funders had no role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

The present study was conducted in conformity with ethical considerations established by the ethical committee of Guangdong Provincial People&#39;s Hospital, Southern Medical University, Guangzhou, China (Approval Number: KY-Q-2022-384-02). Patients in the age range of 18-60 years old were included in this study. Patients taking antibiotics, antibacterial Chinese medicines, drugs such as proton pump inhibitors (PPI), or H2 receptor antagonists, etc., within 2 weeks prior to testing were not included in this study. Those patients who had received treatment against H.pylori in the past 3 months were also excluded from this study. Those with serious heart, liver, kidney problems, severe neuropathy, or mental illness were also not allowed to participate in this study. There were no pregnant women and lactating mothers included in this study. Details of the supplies (reagents, chemicals, equipment, and software) used in this study are given in the Table of Materials.
1. String test for gastric fluid sampling
<ol>
	<li>Ask the patient to fast overnight before the sample collection.</li>
	<li>The next day, open the string test kit, take the capsule, and hold the loop at the end of the string.</li>
	<li>Tape the string onto the patient&#39;s cheek, and ask them to place the capsule on their tongue near the back of the throat and swallow it with a sip of water.</li>
	<li>Allow the capsule to be dissolved in the patient&#39;s stomach, thus exposing the string within the capsule to absorb gastric mucus.</li>
	<li>Ask a trained assistant to pull out the string carefully 1 h after the patient swallowed the capsule.</li>
	<li>Cut off the lower end of the string (40 cm) soaked in the gastric fluid, place it in the TSE (Tris/saline/EDTA) sample preservation solution, and then send it to the clinical laboratory at room temperature (RT) for the subsequent detection of H. pylori and the determination of the antibiotic resistance profiles using qPCR.</li>
</ol>
2. DNA extraction
<ol>
	<li>Place the specimen-carrying collection tubes on a test tube rack, properly labeled, and vortex them for 10 s.</li>
	<li>Turn the 32-well plate (nucleic acid extraction or purification kit) upside down several times to resuspend the magnetic beads. After a short vortex for 10 s, carefully remove the aluminum foil sealing from the plate.</li>
	<li>Transfer 200 &#181;L of gastric fluid from each sample and H. pylori DNA as a positive control into the separate wells.</li>
	<li>Place the 32-well plate in the corresponding sample slot of the nucleic acid extractor machine for automatic DNA extraction.</li>
	<li>After DNA extraction and confirmation, initiate the antibiotic resistance testing for clarithromycin and levofloxacin through qPCR on the positive samples.</li>
	<li>Place the excessive gastric fluid and extracted DNA samples at &#8722;20 &#176;C for long-term storage and future use.</li>
	<li>Perform all these steps in a biosafety cabinet to avoid any contamination.</li>
</ol>
3. qPCR for the detection of H. pylori and resistance to antibiotics (clarithromycin and levofloxacin)
<ol>
	<li>Perform qPCR for the detection of H. pylori and the suspected antibiotic resistance mutations in its genome.
	<ol>
		<li>Thaw the qPCR reaction mixture (H. pylori nucleic acid detection kit) on ice, and mix it by flicking and spinning to prevent any loss of reagent.</li>
		<li>For each sample on a 32-well plate, mix 20 &#181;L of PCR reaction mixture (genotyping reaction premix [containing the Taq enzyme, deoxyribonucleoside triphosphate, reaction buffer, and magnesium chloride], with ureA forward and reverse primers, and a ureA probe), and 5 &#181;L of the extracted DNA.</li>
		<li>Run the 32-well qPCR plate on the qPCR machine. Program the thermal cycler: the reaction mixture will be first reacted sequentially at 42 &#176;C and 95 &#176;C (both for one cycle) for 2 min each; then, 40 cycles at 95 &#176;C, denaturation for 10 s, and annealing and extension at 58 &#176;C for 45 s.</li>
		<li>Set the fluorescence signal acquisition to FAM (H. pylori) and the data acquisition to the amplification extension period. After the reaction is completed, save the data for future results analysis.</li>
		<li>Analyze the data using specific software for qPCR, as the instrument will automatically select baseline thresholds.</li>
		<li>If the test result for H. pylori is positive, the machine will automatically start drug resistance testing on the sample. Replace the reagent kit with detection reagents for 23S rRNA gene and gyrA gene mutations in the H. Pylori (qPCR) kit. 
		NOTE: The reagent kit (detection reagents for 23S rRNA gene and gyrA gene mutations in Helicobacter pylori [qPCR]) includes the PCR reaction mixture (genotyping reaction premix [containing the Taq enzyme, deoxyribonucleoside triphosphate, reaction buffer, magnesium chloride], primers, and probes). All the negative quality control products are sterile, purified water. The positive quality control product in the H. pylori nucleic acid detection kit is inactivated H. pylori standard beads (ATCC 43504). The strong H. pylori-positive and weak positive quality control products in the kit (detection reagents for 23S rRNA gene and gyrA gene mutations in H. pylori) are the DNA of the inactivated H. pylori strain containing the target gene and the mutant gene. Similarly, based on the actual situation, all diagnostic standards, including the presence of H. pylori infection or drug resistance, are set to CT &#8804; 35 and have a typical S-shaped curve. The concentration of the weak quality control is 1.0 x 103 copies/mL, while the concentration of the strong quality control is 1.0 x 108 copies/mL.</li>
	</ol>
	</li>
</ol>

qPCR-Based Rapid, Noninvasive Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

<ol>
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I., Burmistrov, A. I., Maev, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori:+Commensal,+symbiont+or+pathogen.">Helicobacter pylori: Commensal, symbiont or pathogen.</a> World Journal of Gastroenterology. 27 (7), 545-560 (2021).</li><li>Thrift, A. P., Wenker, T. N., El-Serag, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+gastric+cancer:+Epidemiological+trends,+risk+factors,+screening+and+prevention.">Global burden of gastric cancer: Epidemiological trends, risk factors, screening and prevention.</a> Nature Reviews Clinical Oncology. 20 (5), 338-349 (2023).</li><li>Liou, J. 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A., Al-Ouqaili, M. T. S., Majeed, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Helicobacter+pylori+infection+by+invasive+and+noninvasive+techniques+in+patients+with+gastrointestinal+diseases+from+Iraq:+A+validation+study.">Detection of Helicobacter pylori infection by invasive and noninvasive techniques in patients with gastrointestinal diseases from Iraq: A validation study.</a> PLoS One. 16 (8), e0256393 (2021).</li><li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+sensing+strategies+based+on+different+types+of+biomarkers+toward+early+diagnosis+of+H.+pylori.">Advanced sensing strategies based on different types of biomarkers toward early diagnosis of H. pylori.</a> Critical Reviews in Analytical Chemistry. , (2023).</li><li>Hussein, R. A., Al-Ouqaili, M. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+clarithromycin-resistant+Helicobacter+pylori+in+clinical+specimens+by+molecular+methods:+A+review.">Detection of clarithromycin-resistant Helicobacter pylori in clinical specimens by molecular methods: A review.</a> Journal of Global Antimicrobial Resistance. 4, 35-41 (2016).</li><li>Perez-Trallero, E., Montes, M., Alcorta, M., Zubillaga, P., Telleria, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-endoscopic+method+to+obtain+Helicobacter+pylori+for+culture.">Non-endoscopic method to obtain Helicobacter pylori for culture.</a> Lancet. 345 (8950), 622-623 (1995).</li><li>DiNardo, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+string+test+and+stool+specimens+to+diagnose+pulmonary+tuberculosis.">Use of string test and stool specimens to diagnose pulmonary tuberculosis.</a> International Journal of Infectious Diseases. 41, 50-52 (2015).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hypervirulent+Klebsiella+pneumoniae+isolates+using+the+string+test+in+combination+with+Galleria+mellonella+infectivity.">Identification of hypervirulent Klebsiella pneumoniae isolates using the string test in combination with Galleria mellonella infectivity.</a> European Journal of Clinical Microbiology and Infectious Diseases. 39 (9), 1673-1679 (2020).</li><li>Agbonlahor, D. E., Odugbemi, T. O., Udofia, P. 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H. pylori detection can be performed using both invasive and noninvasive methods13. Commonly used invasive techniques such as histopathology, the rapid urease test, polymerase chain reaction (PCR), and bacterial culturing require endoscopy and biopsy.Serological tests, urea breath tests, and enzyme-linked immunosorbent assays (ELISA) are recommended among the noninvasive procedures14. While noninvasive methods are easy to perform, economical, and more comfortable f...

H. pylori is a spiral-shaped, highly motile, gram-negative bacterium that mainly lives in the pylorus region of the stomach1. It&#160;is a common pathogen that infects nearly 50% of the global population2. Most people with H. pylori infection have no clinical manifestations, and most develop different diseases after several years of infection, including chronic gastritis, peptic ulcers, gastric ulcers, and gastric cancer3. In several studies based on different populations, the efficacy of eliminating H. pylori for preventing stomach cancer and precancerous lesions has been demonstrated4,5. Therefore, the World Health Organization (WHO) International Agency for Research on Cancer has advised H. pylori eradication as a preventative measure6.
The use of noninvasive methods to identify H. pylori infection is a key component of treatment for most individuals with asymptomatic dyspepsia. The urea breath test (UBT), H. pylori fecal antigen test (SAT), and serological testing are popular noninvasive techniques. Among these, the UBT is the least intrusive and most accurate procedure available. UBT uses urease, abundantly present in H. pylori, to hydrolyze isotopically labeled urea into ammonia and carbon dioxide (13C or 14C). In contrast, the immunochromatographic assay (ICA)7 is convenient, simple, and noninvasive for sampling. However, the accuracy of the test is affected by several factors, such as the quality of the stool sample, the temperature, and the interval between the sample collection and testing. Another test based on the immune response is the serum H. pylori antibody test, which detects antibodies in a patient&#39;s serum. However, this test is not suitable for post-treatment analysis since the antibodies remain long after the bacteria have been cleared8. Another major drawback is that these methods only diagnose H. pylori infection and do not allow for drug resistance testing to guide sensitivity-based treatment.
For invasive testing methods, gastric biopsy tissue needs to be taken by endoscopy and then subjected to histology, the urease rapid test, and bacterial culture. These testing methods are also very limited due to several factors. Currently, these techniques are limited to elderly patients, patients at high risk for precancerous or malignant disease, and patients who have failed first-line therapy for gastroesophageal reflux disease or H. pylori infection9. Secondly, due to the unique growth characteristics of H. pylori, the success rate of bacterial culture only reaches 50%10. Thus, molecular detection methods offer new hope to overcome the high demands of invasive detection methods and guide sensitivity-based treatment. Among molecular detection methods, quantitative PCR has evolved tremendously in recent years. qPCR, unlike traditional PCR, does not require gel electrophoresis and accurately quantifies DNA/RNA in samples by adding primers and probes at the annealing stage. qPCR kits for the detection of H. pylori infection and drug resistance are now commercially available. Nevertheless, each method has its limitations; therefore, a patient&#39;s clinical diagnosis and treatment should be considered in conjunction with their symptoms, signs, history, other laboratory tests, and response to treatment.
Currently, the primary method of treating H.pylori infections is taking antibiotics, but lately, it is becoming increasingly difficult to treat these infections due to the rise in antibiotic resistance. Subsequently, a significant decline in H. pylori treatment efficacy has been observed globally, making H. pylori eradication a major public health issue11.
Clarithromycin and levofloxacin are the two broad-spectrum antibiotics used to treat infections caused by H.pylori,&#160;but several studies have reported widespread resistance against these two drugs in H.pylori isolates. A2143G, A2142G, and A2142C are three of the numerous point mutations found in the 2.9 kb 23S rRNA gene that result in clarithromycin resistance by preventing the macrolide from binding. At the same time, the mutation loci of the levofloxacin resistance gene are mainly located in the six mutation sites (A260T, C261A, T261G, G271A, G271T, A272G) of the gyrA gene12. The discovery of these resistance mechanisms based on genetic mutations has led to a gradual shift in the detection of H. pylori through cultural-based studies to molecular testing.
Overall, there is an urgent clinical need for a noninvasive, effective, and simultaneous diagnostic method for the detection of H. pylori infections and drug resistance. We adopted a combined string test and qPCR method to overcome the difficulties of sampling and achieve the goal of the simultaneous detection of H. pylori infection and drug resistance using different primer probes.

<table><tbody><tr><td>23S rRNA and gyrA gene point mutations detection kit (PCR-Fluorescence Probing)</td><td>Hongmed Infagen</td><td></td><td>Detection of Helicobacter pylori resistance to clarithromycin and levofloxacin</td></tr><tr><td>ABI 7500 fluorescence quantitative PCR machine</td><td>Thermo Fisher Scientific</td><td>SEDA 20163220767</td><td>Fluorescent quantitative PCR amplification</td></tr><tr><td>ABI 7500 software</td><td>Thermo Fisher Scientific</td><td></td><td>Data Analysis</td></tr><tr><td>BSC-1500IIA2-X</td><td>BIOBASE</td><td>SEDA 20143222263</td><td>Biosafety cabinet</td></tr><tr><td>DNA extraction kit</td><td>Daan Gene&amp;nbsp;</td><td></td><td></td></tr><tr><td>E-Centrifuge</td><td>WEALTEC</td><td></td><td>Centrifuge the residual liquid off the wall of the tube.</td></tr><tr><td>H. Pylori DNA detection kit (PCR-Fluorescence Probing)&amp;nbsp;</td><td>Hongmed Infagen</td><td></td><td>Testing for H. pylori infection</td></tr><tr><td>Stream SP96 automated nucleic acid extractor</td><td>Daan Gene</td><td>SEDA 20140104</td><td>For DNA extraction&amp;nbsp;</td></tr><tr><td>String test kit</td><td>Hongmed Infagen</td><td></td><td>It contains a capsule attached to a string, scissors, cotton swab, and sample preservation tube&amp;nbsp;</td></tr><tr><td>Ultra-low temperature freezers&amp;nbsp;(DW-YL450)&amp;nbsp;</td><td>MELING</td><td>SEDA 20172220091</td><td>-20 &amp;deg;C for storing reagents&amp;nbsp;</td></tr><tr><td>Vortex-5</td><td>Kylin-bell</td><td></td><td>For mixing reagent&amp;nbsp;</td></tr></tbody></table>

quantitative polymerase chain reaction (qpcr)-based rapid diagnosis of helicobacter pylori infection and antibiotic resistance

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to its increasing antibiotic resistance. It is the causative agent of chronic active gastritis, peptic ulcer disease, and gastric cancer and has been classified as a Group I Carcinogen by the International Agency for Research on Cancer. Therefore, the rapid and accurate diagnosis of H. pylori and the determination of its antibiotic resistance are important for the efficient eradication of this bacterial pathogen. Currently, H. pylori diagnosis methods mainly include the urea breath test (UBT), the antigen test, the serum antibody test, gastroscopy, the rapid urease test (RUT), and bacterial culture. Among them, the first three detection methods are noninvasive, meaning they are easy tests to conduct. However, bacteria cannot be retrieved through these techniques; thus, drug resistance testing cannot be performed. The last three are invasive examinations, but they are costly, require high skills, and have the potential to cause damage to patients. Therefore, a noninvasive, rapid, and simultaneous method for H. pylori detection and drug resistance testing is very important for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific procedure involving the string test in combination with quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori infection and antibiotic resistance. Unlike bacterial cultures, this method allows for easy, rapid, noninvasive diagnosis of H. pylori infection status and drug resistance. Specifically, we used qPCR to detect rea for H. pylori infection and mutations in the 23S rRNA and gyrA genes, which encode resistance against clarithromycin and levofloxacin, respectively. Compared to routinely used culturing techniques, this protocol provides a noninvasive, low-cost, and time-saving technique to detect H. pylori infection and determine its antibiotic resistance using qPCR.

Detection of H. pylori infection and antibiotic resistance in stomach fluid by qPCR 
We performed qPCR for the detection of H. pylori infection by amplifying the ureA gene and determined its antibiotic resistance profile by targeting point mutations in the 23S rRNA gene and gyrA gene (Table 1). The quality control CT values in all three groups of the qPCR experiments were within the recommended range, indicating that the samples were all in a norm...

Watch this Scientific Journal Video about Detection and Treatment of Helicobacter pylori Infection at JoVE.com

Author Spotlight: Detection and Treatment of Helicobacter pylori Infection 

The protocol presents a noninvasive method for the rapid diagnosis of Helicobacter pylori stomach infections through the string test and determines its antibiotic resistance to clarithromycin and levofloxacin using quantitative polymerase chain reaction (qPCR).

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to ...

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3.1K Views.  Southern Medical University. Maagkanker veroorzaakt door de maagbacterie, Helicobacter pylori, doodt elk jaar 300.000 mensen in China. Dit onderzoek beschrijft de ingeslikte snaartest waarmee we Helicobacters niet-invasief kunnen vangen en vervolgens testen, zodat patiënten de allerbeste antibioticabehandeling kunnen krijgen. We denken dat elke arts in China gepersonaliseerde, precisiegeneeskunde zal willen beoefenen door deze test te gebruiken om hun behandeling te begeleiden.We richten ons momenteel op de ontwi...

Video: Auteur in de schijnwerpers: detectie en behandeling van Helicobacter pylori-infectie 

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Environment

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Biology

Gastric Mucosa Quantitative Polymerase Chain Reaction Analysis for Detecting Helicobacter pylori and Antibiotic Resistance

Helicobacter pylori is a main pathogen that infects nearly half of the global population and is threatening public health due to its increasing antibiotic resistance. Besides, Helicobacter pylori is also responsible for chronic gastritis, gastric and duodenal ulcers, gastric carcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, it is essential to perform a timely and accurate diagnosis of H. pylori and the determination of its antibiotic resistance. Nowadays, existing methods&#160;of H. pylori diagnosis mainly include the rapid urease test (RUT), the urea breath test (UBT), the serum antibody test, the antigen test, gastroscopy, and bacterial culture. However, bacteria could not be cultured through the first five detection methods, not to mention the detection of drug resistance. The bacterial culture is time-consuming, and antibiotic sensitivity tests cannot be carried out rapidly and routinely. In clinical settings, the swift and precise identification of H. pylori and its susceptibility to antibiotics is crucial for its effective elimination. The objective of this protocol is to outline a targeted approach utilizing quantitative polymerase chain reaction (qPCR) on gastric mucosal samples to expedite the diagnosis of H. pylori and assess its resistance to antimicrobial agents. qPCR was exploited to detect the ureA gene for H. pylori infection and mutations in the 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively. Currently, there remain challenges in gastric mucosa qPCR due to the lack of standard operating procedures. Therefore, it is essential to share methodologies with experimental details to ensure accurate communication of experimental procedures, contributing to gold-standard protocols that enable greater transparency. Overall, this protocol offers an economical and expeditious alternative to conventional methods for assessing H.pylori infection and its resistance to antibiotics through the application of quantitative polymerase chain reaction (qPCR) technology.

A rapid and accurate method for H. pylori detection and drug resistance testing is very significant for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific methodology involving gastric mucosa quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori and antibiotic resistance.

Helicobacter pylori is a main pathogen that infects nearly half of the global population and is threatening public health due to its increasing antibiotic ...

Medicine

Quantification of Helicobacter pylori Load in an Infected Mouse Stomach

To count the number of viable H. pylori in the stomach post-infection, homogenize stomach samples stored in BHI, and perform duplicate serial dilutions of the resulting gastric homogenates in fresh sterile broth.
Divide pre-dried horse blood agar, or HBA plates, supplemented with additional antibiotics into three or four segments, and using an adaptation of the Miles and Misra technique, add 10 to 100 microliters of each stomach homogenate dilution onto a segment of each agar plate. Spread the homogenates with sterile plastic loops and allow the plates to dry. Then, place the plates in an inverted position in anaerobic gas jars containing a Petri dish of water and a gas pack, then, incubate the jars at 37 degrees Celsius for four to seven days.
When colonies can be observed, enumerate the segments containing between 10 and 100 isolated colonies.

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Samenvatting

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