Antimicrobial Resistance Markers Research Articles

Distribution of antimicrobial resistance and virulence markers in chicken originated Proteusmirabilis isolates.

Proteus mirabilis is a common enteric bacterium in livestock and humans. The increase and spread of the antimicrobial resistant P. mirabilis is considered alarming worldwide. Transmission mainly occurs through consumption of contaminated poultry products. We investigated antimicrobial resistance (AMR) and virulence markers in broiler chicken-originated P. mirabilis isolates from 380 fecal samples. Phenotypic AMR test was performed against seventeen different antimicrobials. Genotypic AMR test was performed to detect sixteen different AMR genes. The samples were also tested for the presence of eight different virulence genes and biofilm formation. P. mirabilis was isolated in 11% of the samples, with significantly high multidrug-resistant (MDR) prevalence (63%). All isolates were resistant to tetracycline (100%). The combined disc method indicated that all isolates were of extended-spectrum beta-lactamase (ESBL) producers, which was compatible with the high blaTEM prevalence (95%). This was associated with blaTEM being responsible for more than 80% of ampicillin resistance in enteric pathogens. The absence of phenotypically carbapenem-resistant isolates was compatible with the very low prevalences of blaOXA (2%) and blaNDM (0%). All isolates were positive for pmfA, atfA, hpmA, and zapA (100%) virulence genes, while biofilm formation rate (85%) indicated high adherence abilities of the isolates.

Genetic determinants of resistance to antimicrobial therapeutics are rare in publicly available Clostridioides difficile genome sequences.

To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively. We measured the antimicrobial susceptibility of 143 selected C. difficile isolates. The frequency of mutations causing reduced susceptibility to vancomycin and metronidazole, respectively, increased strongly after 2000, peaking at up to 52% of all sequenced C. difficile genomes. However, both mutations declined sharply more recently, reflecting major changes in CDI epidemiology. We detected mutations associated with fidaxomicin resistance in several major genotypes, but found no evidence of international spread of resistant clones. The pCD-METRO plasmid, conferring metronidazole resistance, was detected in a single previously unreported C. difficile isolate, recovered from a hospital patient in Germany in 2008. The pX18-498 plasmid, putatively associated with decreased vancomycin susceptibility, was confined to related, recent isolates from the USA. Phenotype measurements confirmed that most of those genetic features were useful predictors of antibiotic susceptibility, even though ranges of MICs typically overlapped among isolates with and without specific mutations. Genomic data suggested that resistance to therapeutic antimicrobial drugs is rare in C. difficile. Public antimicrobial resistance marker databases were not equipped to detect most of the genetic determinants relevant to antibiotic therapy of CDI.

Breaking Boundaries in Pneumonia Diagnostics: Transitioning from Tradition to Molecular Frontiers with Multiplex PCR.

The advent of rapid molecular microbiology testing has revolutionized infectious disease diagnostics and is now impacting pneumonia diagnosis and management. Molecular platforms offer highly multiplexed assays for diverse viral and bacterial detection, alongside antimicrobial resistance markers, providing the potential to significantly shape patient care. Despite the superiority in sensitivity and speed, debates continue regarding the clinical role of multiplex molecular testing, notably in comparison to standard methods and distinguishing colonization from infection. Recent guidelines endorse molecular pneumonia panels for enhanced sensitivity and rapidity, but implementation requires addressing methodological differences and ensuring clinical relevance. Diagnostic stewardship should be leveraged to optimize pneumonia testing, emphasizing pre- and post-analytical strategies. Collaboration between clinical microbiologists and bedside providers is essential in developing implementation strategies to maximize the clinical utility of multiplex molecular diagnostics in pneumonia. This narrative review explores these multifaceted issues, examining the current evidence on the clinical performance of multiplex molecular assays in pneumonia, and reflects on lessons learned from previous microbiological advances. Additionally, given the complexity of pneumonia and the sensitivity of molecular diagnostics, diagnostic stewardship is discussed within the context of current literature, including implementation strategies that consider pre-analytical and post-analytical modifications to optimize the clinical utility of advanced technologies like multiplex PCR.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX−M-1 group, blaCTX−M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Antibiotic resistance, plasmids, and virulence-associated markers in human strains of Campylobacter jejuni and Campylobacter coli isolated in Italy.

Campylobacteriosis, a prevalent foodborne gastrointestinal infection in Europe, is primarily caused by Campylobacter jejuni and Campylobacter coli, with rising global concerns over antimicrobial resistance in these species. This study comprehensively investigates 133 human-origin Campylobacter spp. strains (102 C. jejuni and 31 C. coli) collected in Italy from 2013 to 2021. The predominant Multilocus Sequence Typing Clonal complexes (CCs) were ST-21 CC and ST-206 CC in C. jejuni and ST-828 CC in C. coli. Ciprofloxacin and tetracycline resistance, mainly attributed to GyrA (T86I) mutation and tet(O) presence, were prevalent, while erythromycin resistance was associated with 23S rRNA gene mutation (A2075G), particularly in C. coli exhibiting multidrug-resistant pattern CipTE. Notable disparities in virulence factors among strains were observed, with C. jejuni exhibiting a higher abundance compared to C. coli. Notably, specific C. jejuni sequence types, including ST-21, ST-5018, and ST-1263, demonstrated significantly elevated counts of virulence genes. This finding underscores the significance of considering both the species and strain-level variations in virulence factor profiles, shedding light on potential differences in the pathogenicity and clinical outcomes associated with distinct C. jejuni lineages. Campylobacter spp. plasmids were classified into three groups comprising pVir-like and pTet-like plasmids families, exhibiting diversity among Campylobacter spp. The study underscores the importance of early detection through Whole Genome Sequencing to identify potential emergent virulence, resistance/virulence plasmids, and new antimicrobial resistance markers. This approach provides actionable public health data, supporting the development of robust surveillance programs in Italy.

162. The Genotypic Antibiogram: Using Results of Gram-Negative Antimicrobial Resistance Genes Identified via Rapid Blood Culture Identification Tests to Optimize Treatment of Enterobacterales Bloodstream Infections

Abstract Background With the increasing incidence of infections from Enterobacterales resistant to commonly used antibiotics, rapid diagnostic tests, such as BioFire’s Blood Culture Identification 2 (BCID2) multiplex PCR panel, can be utilized to detect potential antimicrobial resistance more rapidly and help direct more appropriate empiric therapy. We describe the epidemiology of Enterobacterales bloodstream infection (BSI), associated antimicrobial resistance genes using BCID2, and subsequent susceptibility patterns at an academic medical center to develop a genotypic antibiogram. Methods We reviewed all positive BCID2 results at Nebraska Medical Center from 8/1/2021–11/1/2022. Only monomicrobial Enterobacterales BSI were included. Patient demographics, antimicrobial resistance markers, BCID2, culture, and susceptibility results were assessed. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of blaCTX-M marker were compared to ceftriaxone susceptibility patterns. Results We reviewed 456 unique Enterobacterales isolates, of which 236 were from male patients (52%), 189 (41%) immunocompromised patients, and 342 (75%) community onset. The most common species identified were Escherichia coli (55%) and Klebsiella pneumoniae (17%). blaCTX-M was detected in 49 (11%) isolates, with 41 (84%) E. coli and 6 (12%) K. pneumoniae. blaKPC was detected in 2 isolates (1 K. oxytoca and 1 K. variicola), but no other carbapenemase genes were detected. blaCTX-M sensitivity and specificity for detection of ceftriaxone resistance was 83% and 100%, respectively. blaCTX-M PPV for ceftriaxone resistance was 100%, while the NPV of absent blaCTX-M for ceftriaxone susceptibility was 97%. Conclusion Locally, the majority of ceftriaxone-resistant E. coli and Klebsiella BSIs harbor blaCTX-M, while carbapenemases are rare. Utilization of rapid diagnostics can optimize antimicrobial therapy for Enterobacterales by creation of a genotypic antibiogram. Disclosures Scott J. Bergman, PharmD, bioMerieux, Inc.: Honoraria Trevor C. Van Schooneveld, MD, FSHEA, FACP, AN2 Therapeutics: Grant/Research Support|Biomeriuex: Advisor/Consultant|Biomeriuex: Grant/Research Support|Insmed: Grant/Research Support|Thermo-Fischer: Honoraria

Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance Genes and Core-Genome Multilocus Sequence Typing of Campylobacter jejuni from Different Sources in Lithuania.

Campylobacter jejuni is known as one of the main causative agents of gastroenteritis in humans worldwide, and the rise of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge of special concern. Whole-genome sequencing (WGS) was used to characterize genetic determinants of AMR in 53 C. jejuni isolates from dairy cattle, broiler products, wild birds, and humans in Lithuania. The WGS-based study revealed 26 C. jejuni AMR markers that conferred resistance to various antimicrobials. Genetic markers associated with resistance to beta-lactamases, tetracycline, and aminoglycosides were found in 79.3%, 28.3%, and 9.4% of C. jejuni isolates, respectively. Additionally, genetic markers associated with multidrug resistance (MDR) were found in 90.6% of C. jejuni isolates. The WGS data analysis revealed that a common mutation in the quinolone resistance-determining region (QRDR) was R285K (854G > A) at 86.8%, followed by A312T (934G > A) at 83% and T86I (257C > T) at 71.7%. The phenotypic resistance analysis performed with the agar dilution method revealed that ciprofloxacin (CIP) (90.6%), ceftriaxone (CRO) (67.9%), and tetracycline (TET) (45.3%) were the predominant AMR patterns. MDR was detected in 41.5% (22/53) of the isolates tested. Fifty-seven virulence genes were identified in all C. jejuni isolates; most of these genes were associated with motility (n = 28) and chemotaxis (n = 10). Additionally, all C. jejuni isolates harbored virulence genes related to adhesion, invasion, LOS, LPS, CPS, transportation, and CDT. In total, 16 sequence types (STs) and 11 clonal complexes (CC) were identified based on core-genome MLST (cgMLST) analysis. The data analysis revealed distinct diversity depending on phenotypic and genotypic antimicrobial resistance of C. jejuni.

Use of genome sequencing to resolve differences in gradient diffusion and agar dilution antimicrobial susceptibility testing performance of Neisseria gonorrhoeae isolates in Alberta, Canada.

Agar dilution is the gold standard method for phenotypic antimicrobial susceptibility testing (AST) for Neisseria gonorrhoeae. However, this method is laborious and requires expertise, so laboratories that perform N. gonorrhoeae AST may choose alternative methods such as disk diffusion and gradient diffusion. In this study, we retrospectively compare the performance of gradient diffusion to agar dilution for 2,394 unique N. gonorrhoeae isolates identified in Alberta from 2017 to 2020 against azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, and tetracycline. Genome sequencing was utilized to resolve discrepancies between AST methods, detect antimicrobial resistance markers, and identify trends between error rates and sequence types (STs) of isolates. Over 90% of N. gonorrhoeae isolates were susceptible to azithromycin, cefixime, and ceftriaxone, whereas decreased susceptibility was observed for ciprofloxacin, penicillin, and tetracycline. Categorical (CA) and essential agreement (EA) was poorest between the two methods for penicillin (CA: 86.02%; EA: 77.69%) and tetracycline (CA: 47.22%; EA: 55.96%); however, the low CA was primarily attributed to minor errors. Antimicrobial agents with errors outside of acceptable limits included azithromycin (very major error: 18.42%; major error: 7.73%) and tetracycline (very major error: 6.17%). Genome sequencing on a subset of isolates resolved 30.3% of the azithromycin major errors and confirmed the azithromycin or tetracycline very major errors. Significant associations between certain STs and error types for azithromycin and tetracycline were also identified. Overall, gradient diffusion compared well to agar dilution for cefixime, ceftriaxone, and ciprofloxacin, and genome sequencing was identified as a useful tool to arbitrate discrepant susceptibility testing results between gradient diffusion and agar dilution for N. gonorrhoeae.

Genomic analysis of almost 8,000 Salmonella genomes reveals drivers and landscape of antimicrobial resistance in China.

Foodborne Salmonella infection remains a major public health concern worldwide. With rising antimicrobial resistance, genomic surveillance is key to tracking outbreaks and monitoring transmission, but there is no comprehensive national surveillance scheme for Salmonella involving humans, food, animals, and the environment in China. Moreover, the association between antimicrobial resistance and climate, social, and economic factors has rarely been investigated. Here, we use 1,962 Salmonella isolates collected from 22 Chinese provinces and add 6,035 publicly available genomes to build a Chinese local Salmonella genome database version 2 (CLSGDB v2) representing 30 Chinese provinces, covering 1905-2022. Using the CLSGDB v2, we mapped the landscape and spatiotemporal dynamics of antimicrobial resistance markers, virulome, and mobilome in Salmonella. We identified 317 mcr positive and 745 azithromycin resistance genes positive Salmonella isolates out of 7,997 isolates. We further uncovered the geographic distribution veil of mcr-1, fosA7, fosA3, mph(A), and blaCTX-M-55 genes in China, all of them resistant to the critically important antimicrobials including colistin, fosfomycin, azithromycin, and the third-generation cephalosporins. Interestingly, economic, climatic, and social factors can drive the rise of antimicrobial resistance was observed. Finally, we release the CLSGDB v2 as an open-access database and thus can assist surveillance studies tracking 164 Salmonella enterica serovars and 295 sequence types across the globe. The CLSGDB v2 is freely available at https://nmdc.cn/clsgdbv2.IMPORTANCEWe established the largest Salmonella genome database from China and presented the landscape and spatiotemporal dynamics of antimicrobial resistance genes. We also found that economic, climatic, and social factors can drive the rise of antimicrobial resistance. The Chinese local Salmonella genome database version 2 was released as an open-access database (https://nmdc.cn/clsgdbv2) and thus can assist surveillance studies across the globe. This database will help inform interventions for AMR, food safety, and public health.

B-149 Next-generation Sequencing Via Targeted Metagenomics for Broad Antimicrobial Resistance Surveillance

Abstract Background Antimicrobial-resistant infections secondary to the overuse of antibiotics are a major public health threat. The standard of care for identifying resistance is antimicrobial susceptibility testing, the phenotypic characterization of minimum inhibitory concentrations (mic) for antibiotics in the presence of bacterial growth, but these methods are labor intensive, have long turnaround times, and face challenges with non-cultivable bacteria. Molecular methods for antimicrobial resistance (AMR) ascertainment are high-throughput, robust, and sensitive and have gained interest as a genotypic complement to traditional phenotypic methods. Since the COVID-19 pandemic, next-generation sequencing has emerged as the leading technology for tracking and surveillance of infectious diseases and antimicrobial resistance, emphasizing the need for flexible sequencing panels that are easily interchangeable and capable of detecting pathogens and AMR genes. Methods In a collaboration between Illumina, a national retail pharmacy, and Aegis Sciences Corporation, residual, self-collected nasal swab samples (n = 4390) submitted for COVID-19 testing from patients across the U.S. were sequenced using the Respiratory Pathogen ID/AMR Panel (RPIP, Illumina – For Research Use Only), a target capture-based next-generation sequencing panel targeting 282 respiratory pathogens and 2097 relevant AMR markers. Sequencing data was analyzed using the Explify RPIP Analysis app (BaseSpace, Illumina) and paired with self-reported patient data submitted via an online questionnaire. Data including pathogens and anti-microbial resistance (AMR) genes detected, age, race, gender, residing state of the patient, collection month, self-reported symptoms, and health risks were collated, imported into Microsoft PowerBI software, and used to create interactive visuals for a surveillance dashboard. Results In this cohort of sequenced specimens, 85.1% (n = 3576) had one or more viral, bacterial, or fungal pathogens present, 41% (n = 1816) had one or more AMR genes present, and 38.4% (n = 1685) had one or more pathogens and AMR genes co-detected. Twenty-six different AMR genes were identified with the most prevalent being Erm 23S ribosomal RNA methyltransferase (65.5%; n = 1115). Erm was identified in samples from all 50 US states and was co-detected with staphylococcus aureus (18.2%; n = 216) and streptococcus pneumoniae (4.9%; n = 57), among others. Following Erm in prevalence, this cohort of samples harbored genes for trimethoprim resistant dihydrofolate reductase (dfr = 27.9%), tetracycline-resistant ribosomal protection protein (tet = 21.0%), and macrolide phosphotransferase (MPH = 14.2%). The data also revealed AMR gene-pathogen co-detections listed as CDC serious threats, such as the mecA gene responsible for methicillin resistance in staphylococcal species. The mecA gene was co-detected in 5.3% (n = 28) of staphylococcus aureus positive samples in this cohort. Finally, although not classified as a serious threat, neuraminidase inhibitor resistance, associated with less susceptibility to anti-virals, was found in 12% (n = 15) of all influenza A (H3N2) infected samples (n = 112). Conclusions Completion of these studies demonstrated that next-generation sequencing using targeted metagenomics with RPIP is a cost effective, high-throughput option for large-scale surveillance of AMR genes in potential respiratory infections.

Clonal expansion and rapid characterization of Klebsiella pneumoniae ST1788, an otherwise uncommon strain spreading in Wales, UK.

A multidrug-resistant strain of Klebsiella pneumoniae (Kp) sequence type (ST) 1788, an otherwise uncommon ST worldwide, was isolated from 65 patients at 11 hospitals and 11 general practices across South and West Wales, UK, between February 2019 and November 2021. A collection of 97 Kp ST1788 isolates (including 94 from Wales) was analysed to investigate the diversity and spread across Wales and to identify molecular marker(s) to aid development of a strain-specific real-time PCR. Whole genome sequencing (WGS) was performed with Illumina technology and the data were used to perform phylogenetic analyses. Pan-genome analysis of further Kp genome collections was used to identify an ST1788-specific gene target; a real-time PCR was then validated against a panel of 314 strains and 218 broth-enriched screening samples. Low genomic diversity was demonstrated amongst the 94 isolates from Wales. Evidence of spread within and across healthcare facilities was found. A yersiniabactin locus and the KL2 capsular locus were identified in 85/94 (90.4 %) and 94/94 (100 %) genomes respectively; bla SHV-232, bla TEM-1, bla CTX-M-15 and bla OXA-1 were simultaneously carried by 86/94 (91.5 %) isolates; 4/94 (4.3 %) isolates also carried bla OXA-48 carbapenemase. Aminoglycoside and fluoroquinolone resistance markers were found in 94/94 (100 %) and 86/94 (91.5 %) isolates respectively. The ST1788-specific real-time PCR was 100 % sensitive and specific. Our analyses demonstrated recent clonal expansion and spread of Kp ST1788 in the community and across healthcare facilities in South and West Wales with isolates carrying well-defined antimicrobial resistance and virulence markers. An ST1788-specific marker was also identified, enabling rapid and reliable preliminary characterization of isolates by real-time PCR. This study confirms the utility of WGS in investigating novel strains and in aiding proactive implementation of molecular tools to assist infection control specialists.

Highly multiplexed targeted sequencing strategy for infectious disease surveillance

BackgroundGlobal efforts to characterize diseases of poverty are hampered by lack of affordable and comprehensive detection platforms, resulting in suboptimal allocation of health care resources and inefficient disease control. Next generation sequencing (NGS) can provide accurate data and high throughput. However, shotgun and metagenome-based NGS approaches are limited by low concentrations of microbial DNA in clinical samples, requirements for tailored sample and library preparations plus extensive bioinformatics analysis. Here, we adapted molecular inversion probes (MIPs) as a cost-effective target enrichment approach to characterize microbial infections from blood samples using short-read sequencing. We designed a probe panel targeting 2 bacterial genera, 21 bacterial and 6 fungi species and 7 antimicrobial resistance markers (AMRs).ResultsOur approach proved to be highly specific to detect down to 1 in a 1000 pathogen DNA targets contained in host DNA. Additionally, we were able to accurately survey pathogens and AMRs in 20 out of 24 samples previously profiled with routine blood culture for sepsis.ConclusionsOverall, our targeted assay identifies microbial pathogens and AMRs with high specificity at high throughput, without the need for extensive sample preparation or bioinformatics analysis, simplifying its application for characterization and surveillance of infectious diseases in medium- to low- resource settings.

Molecular Epidemiology of Antibiotic Resistance among Escherichia coli Isolated from Broiler Chickens Sold at Selected Markets in Dar es Salaam, Tanzania

Unwarranted and improper uses of antibiotics in broiler farms contribute to the challenge of antibiotic resistance, making even previously treatable infections, difficult to treat. We conducted a cross sectional study from November 2021 to May 2022 from broiler chicken markets in four districts of Dar es Salaam to determine the extent of antibiotic resistance among E. coli isolates from broiler chickens. A total of 160 E. coli isolates recovered from cloacal swabs were identified by culture and biochemical tests and confirmed by Polymerase Chain Reaction (PCR) assays. Antimicrobial susceptibility testing, targeting seven classes of antibiotics, was performed by disk diffusion method and eleven representative antimicrobial resistance markers corresponding to each antibiotic class were screened by PCR. The highest resistance was found against trimethoprim (75%) and erythromycin (74.37%), while the most common resistance gene was dfrA1 (74.37%) and blaTEM (73.75%). The study also found a high prevalence of multidrug-resistant isolates (84.4%) from at least three antibiotic classes. The results highlight the significant contribution of poultry farming to the spread of antibiotic resistance, with potential consequences for both farmers and human health. Prompt measures are necessary to protect human and animal health..

Microbiology of Eye Infections at the Massachusetts Eye and Ear: An 8-Year Retrospective Review Combined With Genomic Epidemiology

Ocular bacterial infections are important causes of morbidity and vision loss. Early antimicrobial therapy is necessary to save vision, but their efficacy is increasingly compromised by antimicrobial resistance (AMR). We assessed the etiology of ocular bacterial infections seen at Massachusetts Eye and Ear and investigated the molecular epidemiology and AMR profiles of contemporary isolates. Laboratory investigation. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of leading ocular pathogens and their AMR profiles. A total of 1601 isolates were collected from 2014 to 2021, with Staphylococcus aureus (n=621), coagulase-negative staphylococci (CoNS) (n=234), Pseudomonas aeruginosa (n=213), Enterobacteriaceae (n=167), and Streptococcus pneumoniae (n=95) being the most common. Resistance was high among staphylococci, with methicillin resistance (MR) detected in 28% of S aureus and 39.8% of CoNS isolates. Multidrug resistance (MDR) was frequent among MR staphylococci (MRSA 60%, MRCoNS 76.1%). The population of S aureus isolates consisted mainly of 2 clonal complexes (CCs): CC8 (26.1%) and CC5 (24.1%). CC5 strains carried a variety of AMR markers, resulting in high levels of resistance to first-line therapies. Similarly, the population of ocular Staphylococcus epidermidis was homogenous with most belonging to CC2 (85%), which were commonly MDR (48%). Conversely, ocular S pneumoniae, P aeruginosa, and Enterobacteriaceae were often susceptible to first-line therapies and grouped into highly diverse genetic populations. Our data showed that ocular bacterial infections in our patient population are disproportionately caused by strains that are resistant to clinically relevant antibiotics and are associated with major epidemic genotypes with both community and hospital associations.

A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

Time required for nanopore whole genome sequencing of Neisseria gonorrhoeae for identification of phylogenetic relationships.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing. N. gonorrhoeae strains collected from a London Sexual Health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis. Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%, 95%CI: 98.0-100.0), 502/503 (99.8%, CI:98.9-99.9) and 564/565 (99.8%, CI:99.0-100.0) at 10x, 30x and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION. Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers.

Multiplex Detection of Antimicrobial Resistance Genes for Rapid Antibiotic Guidance of Urinary Tract Infections

Identification of antimicrobial resistance markers in urinary tract infections could provide a more targeted approach in the diagnosis and treatment of UTIs while reducing overall public health burdens. We describe a molecular assay as a diagnostic tool for antibiotic resistance characterization to promote faster diagnosis of antibiotic regimens compared to standard microbiology techniques. Targeted antibiotic usage for pathogenic infections remains a main goal for effective antibiotic treatment protocols and reducing the overall public health burden. Rapid identification of the pathogen(s) causing the infection and harboring the antibiotic resistance gene is also a main area of exploration for antibiotic appropriation and stewardship. Urinary tract infections are a common clinical disease and reservoir for pathogenic infection and the development of antibiotic resistance, especially in hospital- and community-acquired settings. Standard methods require urine culture, which is time consuming and relies on phenotypic characterization. A genetic diagnostic method is warranted for the rapid molecular characterization of antibiotic resistance genes to reduce inappropriate exposure to antibiotics while improving the overall treatment model for urinary tract infections. The purpose of this study is to demonstrate logical viability for real-time molecular diagnostics for early identification, active surveillance and overall targeted antibiotic stratification that is proposed as an in vitro rapid and comprehensive tool for assessing proper antibiotic stewardship in UTIs. Here, we describe a multiplex real-time fluorescence polymerase chain reaction (PCR) for probe-based detection of the top 24 antibiotic resistance genes with targeted relationships to target molecular drug classes and administered antibiotics. Multiplexed analysis based on molecular features enables rapid testing while shifting the diagnostic detection paradigm from monocentric infections towards polymicrobial infections. We utilized 366 samples from the FDA-CDC Antimicrobial Resistance Isolate Bank to test the efficacy of the assay and propose a model to infer the identity of bacterial isolates. We found that, in addition to a high level of accuracy in predicting bacterial genus classification, the assay was mostly in agreement with CDC-tested genotypic and phenotypic results. This study provides evidence for using genetic diagnostic methods, such as multiplex qPCR, in the rapid identification of antibiotic resistance (ABR) genes for the characterization and treatment of urinary tract infections.

Benchmarking microbial DNA enrichment protocols from human intestinal biopsies.

Shotgun metagenomic sequencing is a powerful tool for studying bacterial communities in their natural habitats or sites of infection, without the need for cultivation. However, low microbial signals in metagenomic sequencing can be overwhelmed by host DNA contamination, resulting in decreased sensitivity for microbial read detection. Several commercial kits and other methods have been developed to enrich bacterial sequences; however, these assays have not been tested extensively for human intestinal tissues yet. Therefore, the objective of this study was to assess the effectiveness of various wet-lab and software-based approaches for depleting host DNA from microbiome samples. Four different microbiome DNA enrichment methods, namely the NEBNext Microbiome DNA Enrichment kit, Molzym Ultra-Deep Microbiome Prep, QIAamp DNA Microbiome kit, and Zymo HostZERO microbial DNA kit, were evaluated, along with a software-controlled adaptive sampling (AS) approach by Oxford Nanopore Technologies (ONT) providing microbial signal enrichment by aborting unwanted host DNA sequencing. The NEBNext and QIAamp kits proved to be effective in shotgun metagenomic sequencing studies, as they efficiently reduced host DNA contamination, resulting in 24% and 28% bacterial DNA sequences, respectively, compared to <1% in the AllPrep controls. Additional optimization steps using further detergents and bead-beating steps improved the efficacy of less efficient protocols but not of the QIAamp kit. In contrast, ONT AS increased the overall number of bacterial reads resulting in a better bacterial metagenomic assembly with more bacterial contigs with greater completeness compared to non-AS approaches. Additionally, AS also allowed for the recovery of antimicrobial resistance markers and the identification of plasmids, demonstrating the potential utility of AS for targeted sequencing of microbial signals in complex samples with high amounts of host DNA. However, ONT AS resulted in relevant shifts in the observed bacterial abundance, including 2 to 5 times more Escherichia coli reads. Furthermore, a modest enrichment of Bacteroides fragilis and Bacteroides thetaiotaomicron was also observed with AS. Overall, this study provides insight into the efficacy and limitations of various methods for reducing host DNA contamination in human intestinal samples to improve the utility of metagenomic sequencing.

Genome profiling of uropathogenic E. coli from strictly defined community-acquired UTI in paediatric patients: a multicentric study

BackgroundUrinary tract infection (UTI) in children is a common bacterial infection. The emergence of extended-spectrum beta-lactamases (ESBLs) poses a major challenge against the treatment of uropathogens. We aimed to characterize the E. coli isolates recovered from children with UTI for their resistance profile and circulating sequence types (ST).MethodsChildren (> 1.5–18 years of age) from different community health centres of India with symptoms of UTI were enrolled. Isolates causing significant bacteriuria were identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and tested for antimicrobial susceptibility by the automated system, VITEK-2 (Biomeriux, Durhum, US). Nineteen E. coli isolates (15 ESBL positive and 4 ESBL negative) were sequenced in Oxford Nanopore platform followed by core-genome phylogeny, accessory genome cluster analysis, identification of sequence types, mobile genetic elements, genetic antimicrobial resistance markers. The correlation between detection of antimicrobial resistance genes with phenotypic resistance profiles was also investigated.ResultsEleven percent of children had significant bacteriuria [male:female—1:1, > 50% were 11–18 years of age group]. E. coli was predominant (86%) followed by K. pneumoniae (11%). Susceptibility of E. coli was highest against fosfomycin (100%) followed by carbapenems (90.7%) and nitrofurantoin (88.8%). ST131 (15.8%) and ST167 (10.5%) found as high-risk clones with the presence of plasmid [IncFIB (63.1%), IncFIA (52.6%)], and composite transposon [Tn2680 (46.6%)] in many isolates. Few isolates coharboured multiple beta-lactamases including blaNDM-5 (33.3%), blaOXA-1 (53.3%), blaCTX-M-15 (60%) and blaTEM-4 (60%).ConclusionsThis study highlights horizontal transmission of resistance genes and plasmids in paediatric patients at community centers across the nation harbouring multidrug-resistant genes such as blaNDM-5 and blaCTX-M-15 associated with high-risk clones ST131 and ST167. The data is alarming and emphasizes the need for rapid identification of resistance markers to reduce the spread in community. To our knowledge, this is the first multicentric study targeting paediatric UTI patients from the community setting of India.

Epidemiological and Clinical Features of Streptococcus dysgalactiae ssp. equisimilis stG62647 and Other emm Types in Germany.

(1) Background: Streptococcus dysgalactiae subspecies equisimilis (SDSE) is an important β-hemolytic pathogen historically described as mainly affecting animals. Studies epidemiologically assessing the pathogenicity in the human population in Germany are rare. (2) Methods: the present study combines national surveillance data from 2010 to 2022 with a single-center clinical study conducted from 2016 to 2022, focusing on emm type, Lancefield antigen, antimicrobial resistance, patient characteristics, disease severity, and clinical infection markers. (3) Results: The nationwide reported invasive SDSE infections suggest an increasing infection burden for the German population. One particular emm type, stG62647, increased over the study period, being the dominant type in both study cohorts, suggesting a mutation-driven outbreak of a virulent clone. The patient data show that men were more affected than women, although in the single-center cohort, this trend was reversed for patients with stG62647 SDSE. Men affected by stG62647 developed predominantly fascial infections, whereas women suffering from superficial and fascial non-stG62647 SDSE infections were significantly younger than other patients. Increasing age was a general risk factor for invasive SDSE infections. (4) Conclusions: further studies are needed to further elucidate the raised questions regarding outbreak origin, underlying molecular mechanisms as well as sex-dependent pathogen adaptation.