Detection Of Resistance Genes Research Articles

Antimicrobial resistance determinants in the oropharyngeal microbiome of 'men having sex with men' attending an sexually transmitted infection clinic.

'Men having sex with men' (MSM) represent a key population with a significant prevalence of pharyngeal Neisseria gonorrhoeae (NG) infections and a high rate of antimicrobial resistance genes in the pharyngeal microbiome. As NG can acquire antibiotic resistance genes from other commensal oropharyngeal bacteria, monitoring the prevalence of these resistance determinants is critical to curtail the spread of NG-resistant strains. Here, we assessed the distribution of five resistance genes (pen (A), mtr (R), gyr (A), par (C), msr (D)) in the oropharynx of 164 MSM, attending an Outpatient clinic for STI screening. The most frequently detected resistance gene was msr (D) (88.4%), followed by gyr (A) (67.1%). The distribution of resistance genes was not influenced by pharyngeal gonorrhea nor by the HIV status, whereas a younger age was associated with mtr (R) presence (p = .008). Subjects using mouthwash exhibited significantly lower levels of mtr (R) (p = .0005). Smoking habit was associated with a higher prevalence of par (C) (p = .02). A noteworthy association was observed between the presence of msr (D) gene and the use of antibiotics (p = .014). Our findings reveal an enrichment of antimicrobial resistance genes in the oropharynx of MSM. These insights could aid in the development of screening programs and antimicrobial stewardship initiatives targeting populations at heightened risk of pharyngeal gonorrhea.

Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs’ biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.

High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan three times a month between August 2022 and July 2023 were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥ 0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection rates and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.

Dental plaque microbiota sequence counts for microbial profiling and resistance genes detection

Shotgun metagenomics sequencing experiments are finding a wide range of applications. Nonetheless, there are still limited guidelines regarding the number of sequences needed to acquire meaningful information for taxonomic profiling and antimicrobial resistance gene (ARG) identification. In this study, we explored this issue in the context of oral microbiota by sequencing with a very high number of sequences (~ 100 million), four human plaque samples, and one microbial community standard and by evaluating the performance of microbial identification and ARGs detection through a downsampling procedure. When investigating the impact of a decreasing number of sequences on quantitative taxonomic profiling in the microbial community standard datasets, we found some discrepancies in the identified microbial species and their abundances when compared to the expected ones. Such differences were consistent throughout downsampling, suggesting their link to taxonomic profiling methods limitations. Overall, results showed that the number of sequences has a great impact on metagenomic samples at the qualitative (i.e., presence/absence) level in terms of loss of information, especially in experiments having less than 40 million reads, whereas abundance estimation was minimally affected, with only slight variations observed in low-abundance species. The presence of ARGs was also assessed: a total of 133 ARGs were identified. Notably, 23% of them inconsistently resulted as present or absent across downsampling datasets of the same sample. Moreover, over half of ARGs were lost in datasets having less than 20 million reads. This study highlights the importance of carefully considering sequencing aspects and suggests some guidelines for designing shotgun metagenomics experiments with the final goal of maximizing oral microbiome analyses. Our findings suggest varying optimized sequence numbers according to different study aims: 40 million for microbiota profiling, 50 million for low-abundance species detection, and 20 million for ARG identification.Key points• Forty million sequences are a cost-efficient solution for microbiota profiling• Fifty million sequences allow low-abundance species detection• Twenty million sequences are recommended for ARG identificationGraphical

Detection of Vancomycin Resistant Genes in Intrinsically Antibiotic Resistant Bacteria from the Gut Microbiota of Indonesian Individuals.

Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR). The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.

Extended-spectrum β-lactamase-producing bacteria and their resistance determinants in different wastewaters and rivers in Nepal

Wastewaters serve as significant reservoirs of antibiotic resistant bacteria. Despite the evidence of antimicrobial resistance in wastewaters and river water in Kathmandu, direct linkage between them is not discussed yet. This study investigated the prevalence of extended-spectrum β-lactamase (ESBL)-producing bacteria and associated resistance genes in wastewaters and river water. Out of 246 bacteria from wastewaters, 57.72% were ESBL producers and 77.64% of them were multidrug resistant (MDR). ESBL producing E. coli was dominant in municipal and hospital wastewaters (HWW) as well as in river water while K. pneumoniae in pharmaceutical wastewater. The, blaSHV and blaTEM genes were prevalent and commonly co-occurred with aac(6')-Ib-cr in K. pneumoniae isolated pharmaceutical wastewater. blaCTX-M carrying E. coli from hospital co-harbored aac(6')-Ib-cr while that from municipal influent and river with co-harbored qnrS. Whole genome sequencing data revealed the presence of diverse ARGs in bacterial isolates against multiple antibiotics. In average, an E. coli and a K. pneumoniae isolate contained 55.75±0.96 and 40.2±5.36 ARGs. Multi-locus sequence typing showed the presence of globally high-risk clones with wider host range such as E. coli ST10, and K. pneumoniae ST15 and ST307 in HWW and river indicating frequent dissemination of antimicrobial resistance in wastewater of Kathmandu. Whole genome sequence data aligned with phenotypic antibiograms and PCR detected resistance genes in selected isolates. The presence of significant plasmid replicons (IncF, IncY) and mobile genetic elements (IS903, IS26) indicate high frequency of spreading antibiotic resistance. These findings indicate burden and dissemination of antimicrobial resistance in the environment and highlight the need for effective strategies to mitigate the antibiotic resistance. Environmental implicationThe antibiotic resistant bacteria with their resistance marker genes in the environment promote the transfer and proliferation of high degree of resistance. Since this study found that potential pathogenic bacteria in wastewater and river were highly resistant phenotypes and genotypes with significant sequence types containing multiple plasmid replicons and mobile genetic elements indicating severe biological pollution in Kathmandu. These findings have three key environmental implications- 1) high risk of water borne transmission of antibiotic resistant pathogens to humans and animals posing significant public health risks, 2) possible significantly higher spread of antibiotic resistance among or between environmental and clinical pathogens, and 3) identifying critical routes of ARG dissemination and establishing dependable methods for monitoring and evaluating risks, and employing technological innovations to prevent the pollution of the environment with bacteria and genes that possess resistance to antibiotics. This study also provides clues that treatment of hospital wastewater before discharging to rivers could be a notable action to mitigate the associated risks from the environment.

Monitoring and analysis of pathogenic microorganisms and resistance genes.

The objectives are to improve the rapid identification method of microbial risk and cut off the route of transmission of resistance genes. When new pathogenic microorganisms are found, intervention can be carried out as early as possible to identify the risk of potential pathogen transmission, and timely cut off the transmission route. Hospital air samples were collected to analyse the distribution of environmental pathogenic microorganisms and the characteristics of ARGs resistance genes. The air samples were collected from 12 sampling sites in the Affiliated Hospital of Yangzhou University. In the infusion room, general ward and intensive care unit, no significant difference was found in microorganisms, and no significant difference was found in microbial resistance genes. There were some differences in resistance genes between east and west districts. Combined with the detection of pathogenic microorganisms and resistance genes in our hospital, it is necessary to improve the daily disinfection measures such as air conditioning and fresh air equipment, cut off the infection route, block the transmission of resistance genes, and monitor pathogens and resistance genes in airborne diseases.

Molecular Detection of Vancomycin Resistance Genes in Staph aureus Isolates from Different Clinical Specimens

The study screened the occurrence of VanA, VanB, Vanc genes in Staph aureus vancomycin-resistant isolates recovered in Hilla -hospitals. From various clinical samples, 160 isolates from patients between July and November of 2022 were included in the search. Among these samples are (65) burn samples, (55) urine samples, and (40) samples from Blood. 40 (or 25%) of the 160 isolates were Staph aureus. Characterization of the VanA, VanB, and VanC genes in Staph aureus isolates obtained from various specimens is the main goal of the work. The RESULTS of samples were obtained from a variety of clinical specimens, including 20 (40.625%) burn swabs, 10 (25%) urine samples, and 10 (34.375%) blood. Staph aureus was resistant to Cefepime (91.7%), Piperacillin (73.3%), and Ceftazidime (71.7%), although little relationship was detected with Aztreonam resistance (35%). 40 isolates were used for antibiotic sensitivity test (AST). Only 18.3% and 15% of isolates, respectively, were resistant to meropenem and imipenem. Fluoroquinolone resistance to lomefloxacin, ciprofloxacin, ofloxacin, and levofloxacin was 51.7%, 48.3%, 35%), and 30%, respectively. While the most prevalent vancomycin resistance genes were found in 40 isolates of S. aureus and were discovered by VanC 40 (100%), VanA 20 (100%), and VanB28 (70%), according to the current investigation. In Conclusion :The PCR-reaction method was discovered to be an easy-to-use tool for detecting staph aureus by pcr that can be regularly used for identifying staph aureus and detection the molecular resistance apparatuses to vancomycin in staph aureus by VanA, VanB, VanC), efflux systems plays To choose the best antibiotics, prevent the establishment of resistant strains, and create novel antimicrobial. medications, it is beneficial to research the particular resistance mechanisms vancomycin to staph aureus.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

Rapid and sensitive parallel on-site detection of antibiotics and resistance genes in aquatic environments using evanescent wave dual-color fluorescence fiber-embedded optofluidic nanochip

Environmental antibiotics and antibiotic resistance genes (ARGs) pose considerable threat to humans and animals; thus, the rapid and sensitive parallel detection of these pollutants from a single sample is urgently required. However, traditional multiplexed analytic technologies detect only one type of target (e.g., small molecules or nucleic acids) per assay. To address this issue, Evanescent wave Dual-color fluorescence Fiber-embedded Optofluidic Nanochip (EDFON) was fabricated by integrating a fiber-embedded optofluidic nanochip with evanescent wave dual-color fluorescence technology. The EDFON was used for the parallel quantitative detection of sulfamerazine (SMR) and MCR-1 with high sensitivity and specificity by combining a heterogeneous immunoassay with a homogenous hybridization chain reaction based on time-resolved effects. LODs of 0.032 μg/L and 35 pM was obtained for SMR and MCR-1, respectively, within 20 min. To our best knowledge, the EDFON is the first device for the simultaneous detection of two type of targets in each test, which is highly valuable to prevent the global threats of antibiotics and ARGs. Comparison with liquid chromatography-mass spectrometry showed a strong linear relationship (R2 = 0.998) for SMR pollution in the Qinghe River, with spiked SMR and MCR-1 negative surface and wastewater samples showing recovery rates of 91.8–113.4%. These results demonstrate the excellent accuracy and reliability of the EDFON, with features such as multi-analyte detection, field-deployment, and minimal-equipment, rendering it revolutionary for environmental monitoring, food safety, and medical diagnostics.

Antimicrobial resistance prediction by clinical metagenomics in pediatric severe pneumonia patients

BackgroundAntimicrobial resistance (AMR) is a major threat to children’s health, particularly in respiratory infections. Accurate identification of pathogens and AMR is crucial for targeted antibiotic treatment. Metagenomic next-generation sequencing (mNGS) shows promise in directly detecting microorganisms and resistance genes in clinical samples. However, the accuracy of AMR prediction through mNGS testing needs further investigation for practical clinical decision-making.MethodsWe aimed to evaluate the performance of mNGS in predicting AMR for severe pneumonia in pediatric patients. We conducted a retrospective analysis at a tertiary hospital from May 2022 to May 2023. Simultaneous mNGS and culture were performed on bronchoalveolar lavage fluid samples obtained from pediatric patients with severe pneumonia. By comparing the results of mNGS detection of microorganisms and antibiotic resistance genes with those of culture, sensitivity, specificity, positive predictive value, and negative predictive value were calculated.ResultsmNGS detected bacterial in 71.7% cases (86/120), significantly higher than culture (58/120, 48.3%). Compared to culture, mNGS demonstrated a sensitivity of 96.6% and a specificity of 51.6% in detecting pathogenic microorganisms. Phenotypic susceptibility testing (PST) of 19 antibiotics revealed significant variations in antibiotics resistance rates among different bacteria. Sensitivity prediction of mNGS for carbapenem resistance was higher than penicillins and cephalosporin (67.74% vs. 28.57%, 46.15%), while specificity showed no significant difference (85.71%, 75.00%, 75.00%). mNGS also showed a high sensitivity of 94.74% in predicting carbapenem resistance in Acinetobacter baumannii.ConclusionsmNGS exhibits variable predictive performance among different pathogens and antibiotics, indicating its potential as a supplementary tool to conventional PST. However, mNGS currently cannot replace conventional PST.

Antimicrobial Resistance Profiles of Enterococcus Faecalis and Enterococcus Faecium Isolated from Clinical Specimens at Kitwe Teaching Hospital in Zambia

This research examined the occurrence of Enterococcus faecalis and Enterococcus faecium in clinical samples processed at Kitwe Teaching Hospital Laboratory, along with their resistance to antimicrobials and presence of virulence determinants. A total of 230 clinical specimens were analyzed to identify suspected Enterococcus strains. Standard laboratory techniques were employed to isolate, characterize, and confirm the species of Enterococcus, followed by testing their susceptibility to antimicrobials and detection of resistance and virulence genes using polymerase chain reaction. Among the 230 cultured specimens, 89 were stool samples, 3 were swabs, and 138 were urine specimens. Out of these, 71 isolates were confirmed Enterococcus by PCR using genus specific primers. The resulting prevalence rate of 30.9% was thus obtained. The most prevalent species was Enterococcus faecalis, accounting for 56.3% of the 71 Enterococcus isolates. The prevalence of E. faecium was 14.1%. All tested E. faecalis isolates displayed resistance to ciprofloxacin and erythromycin. Furthermore, high rates of resistance were observed among E. faecalis isolates for chloramphenicol and tetracycline (97.5%), vancomycin (90.0%), nitrofurantoin (75.0%), penicillin (75.0%) and ampicillin (67.5%). Only two E. faecium isolates exhibited susceptibility to tetracycline and vancomycin, while the remaining isolates were 100% resistant to all other tested antimicrobials. Multidrug resistance was detected in all E. faecium isolates. In addition, various antibiotic resistance genes (aac(6′)-Ie-aph(2″)-LA, ermA, ermB, tetK, tetL, tetM, tetX, vanA) were identified in both E. faecalis and E. faecium isolates. A positive association between phenotype and genotype was found for tetracycline and erythromycin.

Monitoring of Klebsiella pneumoniae as one of the criteria of microecological characterization of a water body

Introduction. Representatives of the family Enterobacteriaceae are ubiquitous in the environment, causing severe, including opportunistic diseases in immunocompromised individuals. The occurrence of antibiotic-sensitive bacteria of the Klebsiella genus within the recreational zone of a water body is potent of posing an epidemiologic danger to the inhabitants of the settlement. 
 Materials and Methods. Water samples from the surface aquifers of the Don and Temernik rivers of Rostov-on-Don served as the material for the study. The sensitivity of isolates of Kl. pneumoniae isolates to fourteen antibacterial preparations were investigated by disk-diffusion method in accordance with the requirements of MUK 4.2.1980-04 and EUCAST v. 13.0 (2023). 13.0 (2023). Carbapenemase genes VIM, IMP, NDM, KPC, OXA-48, NDM were determined using commercial diagnostic kits AmpliSense MDR MBL-FL, AmpliSense MDR KPC/OXA-48-FL, AmpliSense MDR Ab-OXA-FL.
 Results. In 2022, 247 isolates of Klebsiella pneumoniae were isolated in 14 biotopes of water bodies of Rostov-on-Don. Biotopes of epidemiological significance were identified with the presence of antibiotic resistance genes in 28 isolates isolated above and below the sewage discharge site (sampling point #1 and #5) and strains isolated from the mouth of the Temernik River (sampling point #14). Phenotype of multidrug-resistant (MDR) in point No. 1 possessed 22%, point No. 5 – 26.9%, point No. 14 – 26%; phenotype of extreme drug resistance (XDR) in point No. 1 possessed 11.1 %, point No. 5 – 15.3%, point No. 14 – 13%; phenotype of pan-drug-resistant (PDR) in point No. 1 did not possess, point No. 5 – 3.8%, point No. 14 – 4.3%.
 Limitations. A limitation of the study is related to the location of water sampling from the river. Don and R. Temernik on the territory of Rostov-on-Don, covering from March to November 2022. In this article, we limited ourselves to sanitary-bacteriological and molecular genetic analysis.
 Conclusion. The detection of PDR, XDR, and MDR antibiotic-resistant phenotypes of Klebsiella pneumoniae, as well as the detection of resistance genes, indicates to an increased risk of water-associated acute intestinal infections, especially in immunosuppressed individuals.

Detection of Macrolide and/or Fluoroquinolone Resistance Genes in Mycoplasma genitalium Strains Isolated from Men in the Northwest Region of Croatia in 2018-2023.

Mycoplasma genitalium (M. genitalium) poses a significant public health challenge due to its association with non-gonococcal urethritis (particularly in men) and antimicrobial resistance. However, despite the prevalence of M. genitalium infections and the rise in resistance rates, routine testing and surveillance remain limited. This is the first study from Croatia that aimed to assess the prevalence and trends of resistance in M. genitalium strains isolated from male individuals by detecting macrolide and fluoroquinolone resistance genes. The study also aimed to explore the factors associated with resistance and changes in resistance patterns over time. Urine samples collected from male individuals in the Zagreb County and northwest region of Croatia between 2018 and 2023 were tested for M. genitalium with the use of molecular methods. Positive samples were subjected to DNA extraction and multiplex tandem polymerase chain reaction (MT-PCR) targeting genetic mutations associated with macrolide (23S rRNA gene) and fluoroquinolone (parC gene) resistance. Of the 8073 urine samples tested from 6480 male individuals (and following the exclusion of repeated specimens), we found that the prevalence of M. genitalium infection was 2.2%. Macrolide resistance was observed in 60.4% of strains, while fluoroquinolone resistance was found in 19.2%. Co-resistance to both antibiotics was present in 18.2% of cases. A statistically significant increase in fluoroquinolone resistance was noted over the study period (p = 0.010), but this was not evident for azithromycin resistance (p = 0.165). There were no statistically significant differences in resistance patterns between age groups, whereas re-testing of patients revealed dynamic changes in resistance profiles over time. The high burden of macrolide resistance and increasing fluoroquinolone resistance underscore the urgent need for comprehensive resistance testing and surveillance programs. The implementation of resistance-guided treatment strategies, along with enhanced access to molecular diagnostics, is pivotal for effectively managing M. genitalium infections.

Molecular detection of antimicrobial resistance genes in multidrug-resistant Gram-negative bacteria isolated from clinical samples in two hospitals in Niger

Background: According to the World Health Organization (WHO), bacterial resistance to antibiotics is a global public health challenge, which is also developing in Niger. The aim of this study was to determine the prevalence of antibiotic resistance genes in Gram-negative bacilli isolated from clinical samples in the biological laboratories of two selected health facilities in Niger.
 Methodology: Clinical bacterial isolates were randomly collected from two biological laboratories of Zinder National Hospital and Niamey General Reference Hospital. These were multi-resistant Gram-negative bacteria that have been routinely isolated from pathological samples of patients. Molecular detection of resistance genes was carried out by polymerase chain reaction (PCR) amplification using specific primers. These include plasmid-mediated AmpC beta lactamase genes (blaCITM, blaDHAM, blaFOXM), ‘Cefotaxime-Munich’ type beta lactamase genes (blaCTX-M-1, blaCTX-M-2, blaCTX-M-9), KPC-type beta lactamase gene (blaKPC), Oxa-type beta lactamase gene (blaOXA-48), SHV-type beta lactamase gene (blaSHV), TEM-type beta lactamase gene (blaTEM), quinolone resistance genes (qnrA, qnrB, qnrS), and sulfonamide resistance genes (sul1, sul2, sul3).
 Results: A total of 24 strains of multidrug-resistant Gram-negative bacteria isolated from different clinical samples were analysed. The distribution of the resistance genes detected is as follows; AmpC blaCITM (n=6; 25.0%), AmpC blaDHAM (n=4; 17.0%), AmpC blaFOXM (n=0), blaCTX-M-1 (n=11; 46.0%), blaCTX-M-2 (n=0), blaCTX-M-9 (n=0), blaKPC (n=0), blaOXA-48 (n=2; 8..0%), blaSHV (n=5; 21.0%), blaTEM (n=0), qnrA (n=0), qnrB (n=5; 21.0%), qnrS (n=17; 71.0%), sul1 (n=22; 92.0%), sul2 (n=12; 50.0%), and sul3 (n=0). All isolates tested had at least two resistance genes.
 Conclusion: The results of this study provide a better understanding of the resistance situation of clinical isolates in Niger. Therefore, it is more than necessary to intensify the detection on a larger number of samples and on a national scale. This will make it possible to assess the true extent of the phenomenon and consequently guide control strategies through a national multisectoral plan.
 Contexte: Selon l'Organisation Mondiale de la Santé (OMS), la résistance bactérienne aux antibiotiques constitue un défi mondial de santé publique, qui se développe également au Niger. Le but de cette étude était de déterminer la prévalence des gènes de résistance aux antibiotiques chez les bacilles Gram négatif isolés à partir d'échantillons cliniques dans les laboratoires de biologie de deux formations sanitaires sélectionnées au Niger.
 Méthodologie: Des isolats bactériens cliniques ont été collectés de manière aléatoire dans deux laboratoires de biologie de l'Hôpital National de Zinder et de l'Hôpital Général de Référence de Niamey. Il s’agissait de bactéries Gram-négatives multirésistantes qui ont été systématiquement isolées à partir d’échantillons pathologiques de patients. La détection moléculaire des gènes de résistance a été réalisée par amplification par réaction en chaîne par polymérase (PCR) à l'aide d'amorces spécifiques. Il s'agit notamment des gènes de bêta-lactamase AmpC à médiation plasmidique (blaCITM, blaDHAM, blaFOXM), des gènes de bêta-lactamase de type «Céfotaxime-Munich» (blaCTX-M-1, blaCTX-M-2, blaCTX-M-9), du gène de bêta-lactamase de type KPC (blaKPC), du gène de bêta-lactamase de type Oxa (blaOXA-48), le gène bêta-lactamase de type SHV (blaSHV), le gène bêta-lactamase de type TEM (blaTEM), les gènes de résistance aux quinolones (qnrA, qnrB, qnrS) et les gènes de résistance aux sulfamides (sul1, sul2, sul3).
 Résultats: Au total, 24 souches de bactéries Gram-négatives multirésistantes isolées de différents échantillons cliniques ont été analysées. La répartition des gènes de résistance détectés est la suivante; AmpC blaCITM (n=6; 25,0%), AmpC blaDHAM (n=4; 17,0%), AmpC blaFOXM (n=0), blaCTX-M-1 (n=11; 46,0%), blaCTX-M-2 (n=0), blaCTX-M-9 (n=0), blaKPC (n=0), blaOXA-48 (n=2; 8,0%), blaSHV (n=5; 21,0%), blaTEM (n=0), qnrA (n=0), qnrB (n=5; 21,0%), qnrS (n=17; 71,0%), sul1 (n=22; 92,0%), sul2 (n=12; 50,0%) et sul3 (n=0). Tous les isolats testés possédaient au moins deux gènes de résistance.
 Conclusion: Les résultats de cette étude permettent de mieux comprendre la situation de résistance des isolats cliniques au Niger. Il est donc plus que nécessaire d’intensifier la détection sur un plus grand nombre d’échantillons et à l’échelle nationale. Cela permettra d'évaluer l'ampleur réelle du phénomène et par conséquent d'orienter les stratégies de lutte à travers un plan national multisectoriel.

Antibiotic-Resistance Profiles and Genetic Diversity of Shigella Isolates in China: Implications for Control Strategies.

The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.

Biofilm-producing and specific antibiotic resistance genes in Pseudomonas aeruginosa isolated from patients admitted to a tertiary care hospital, Bangladesh

AimsBiofilms are responsible for persistent infections and antimicrobial resistance. Pseudomonas aeruginosa was investigated with its ability to form biofilm by detecting genes responsible to produce biofilms and biofilm-specific antimicrobial resistance. The association between antibiotic resistance and biofilm was investigated. MethodsThis cross-sectional study was conducted from July 2017 to December 2018. A total of 446 samples (infected burn, surgical wounds, and ETA) were collected from admitted patients of Dhaka Medical College and Hospital, Bangladesh. P. aeruginosa was isolated and identified by biochemical tests and PCR. Biofilm production by Tissue culture plate (TCP) method followed by detection of biofilm-producing genes (pqsA, pslA, pslD, pslH, pelA, lasR) and biofilm-specific antibiotic resistance genes (ndvB, PA1874, PA1876, PA1877) by PCR were done. Antibiotic susceptibility test was carried out by disc diffusion method; for colistin agar dilution method of MIC was followed. ResultsAmong 232 (52.02%) positive strains of P. aeruginosa, 24 (10.30%) produced biofilms in TCPM. Among biofilm-producing genes, pqsA was the highest (79.17%). pslA and pelA were 70.83%, pslD 45.83%, pslH and lasR 37.5%. Among biofilm-specific antibiotic resistance genes, 16.67% were ndvB, and 8.33% were PA1874 and PA1877. Biofilm-forming strains were significantly resistant to colistin. ConclusionDetection of biofilm-forming genes may be a good tool for the evaluation of biofilm production, which will help in prompt and better management of chronic or device-associated infections.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

First report of multidrug-resistant carbapenemase-producing Aeromonas caviae co-harboring mcr-3.43 and mcr-7.2.

Hospital sewage serves as a crucial reservoir for antibiotic resistance genes. As colistin and carbapenems are the last-resort antibiotics, the emergence of their resistance genes has become a significant concern in clinical settings. In this study, we found that two novel mcr alleles (mcr-3.43 and mcr-7.2) with two carbapenemase genes (blaNDM-1 and blaKPC-2) were encoded in a single Aeromonas caviae strain isolated from hospital sewage. Our phylogenetic analysis revealed that the mcr-3.43 gene clustered with mcr-3.17 (with 95.55% amino acid identity), while the mcr-7.2 gene clustered with mcr-7.1 (with 68.68% amino acid identity). BLAST search against GenBank showed that mcr-7.2 was exclusively detected in Aeromonas spp. Mobile genetic elements were not found in the genetic context of mcr-7.2, suggesting that the dissemination of mcr-7.2 in Aeromonas spp. may be dependent on vertical transfer or recombination. The blaNDM-1 was adjacent to a recombinase gene and flanked by two IS91 elements, indicating a potential mobilization mechanism mediated by recombination and/or ISs. The blaKPC-2 gene was located on an IncU plasmid and adjacent to an ISKpn6. In summary, our study provides evidence for Aeromonas spp. as one of the potential reservoirs of colistin and carbapenem resistance genes.IMPORTANCEThe study discovered two novel mcr genes (mcr-3.43 and mcr-7.2) and two carbapenemase genes (blaNDM-1 and blaKPC-2) in a single Aeromonas caviae strain retrieved from hospital sewage. Using phylogenetic analysis and comparative data evaluation, the study revealed the genetic relatedness and dissemination potential of the detected resistance genes. With the exclusive discovery that mcr-7.2 is only present in Aeromonas spp. and the lack of mobile genetic elements in its genetic context, there is a strong indication of limited dissemination. The identification of these four resistance genes in a single strain of Aeromonas provided valuable insights into their potential presence in this genus. This study revealed that hospital sewage functions as a significant reservoir for antibiotic resistance genes, including colistin and carbapenem resistance genes.

Antimicrobial resistance in wildlife: detection of antimicrobial resistance genes in Apennine wolves (Canis lupus italicus Altobello, 1921) from Central Italy.

The aim of this study was to molecularly investigate the presence of antimicrobial resistance genes (ARGs) in organ samples from 11 Apennine wolves (Canis lupus italicus) collected in Central Italy. Samples from lung, liver, spleen, kidney, tongue and intestine were investigated by PCRs targeting the following genes: tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tetA(P), tet(Q), tet(S), tet(X), sul1, sul2, sul3, blaCTX-M, blaSHV, blaTEM and mcr-1. A PCR positivity was highlighted for 13 out of the 21 tested genes; no positive results were obtained for tet(C), tet(D), tet(E), tet(G), sul3, blaCTX, blaSHV and mcr-1 genes. All 11 animals sampled showed positivity for one or more resistance genes. The results confirm the potential role of the wolf as an indicator and/or vector of antimicrobial-resistant bacteria or ARGs.