Antimicrobial Resistance Genes Research Articles

Multi-omics strategy reveals potential role of antimicrobial resistance and virulence factor genes responsible for Simmental diarrheic calves caused by Escherichia coli.

Escherichia coli (E. coli) is reported to be an important pathogen associated with calf diarrhea. Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) pose a considerable threat to both animal and human health. However, little is known about the characterization of ARGs and VFGs presented in the gut microbiota of diarrheic calves caused by E. coli. In this study, we used multi-omics strategy to analyze the ARG and VFG profiles of Simmental calves with diarrhea caused by E. coli K99. We found that gut bacterial composition and their microbiome metabolic functions varied greatly in diarrheic calves compared to healthy calves. In total, 175 ARGs were identified, and diarrheal calves showed a significantly higher diversity and abundance of ARGs than healthy calves. Simmental calves with diarrhea showed higher association of VFGs with pili function, curli assembly, and ferrienterobactin transport of E. coli. Co-occurrence patterns based on Pearson correlation analysis revealed that E. coli had a highly significant (P < 0.0001) correlation coefficient (>0.8) with 16 ARGs and 7 VFGs. Metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Phylotype analysis of E. coli genomes showed that the predominant phylogroup B1 in diarrheic Simmental calves was associated with 10 ARGs and 3 VFGs. These findings provide an overview of the diversity and abundance of the gut microbiota in diarrheic calves caused by E. coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the calves affected with diarrhea.IMPORTANCESimmental is a well-recognized beef cattle breed worldwide. They also suffer significant economic losses due to diarrhea. In this study, fecal metagenomic analysis was applied to characterize the antibiotic resistance gene (ARG) and virulence factor gene (VFG) profiles of diarrheic Simmental calves. We identified key ARGs and VFGs correlated with Escherichia coli isolated from Simmental calves. Additionally, metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Our findings provide an insight into the diversity and abundance of the gut microbiota in diarrheic calves caused by Escherichia coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the diarrheal calves from cattle hosts.

WGS of intrauterine E. coli from cows with early postpartum uterine infection reveals a non-uterine specific genotype and virulence factors.

Escherichia coli has been attributed to playing a major role in a cascade of events that affect the prevalence and severity of uterine disease in cattle. The objectives of this project were to (i) define the association between the prevalence of specific antimicrobial resistance and virulence factor genes in E. coli with the clinical status related to uterine infection, (ii) identify the genetic relationship between E. coli isolates from cows with diarrhea, with mastitis, and with and without metritis, and (iii) determine the association between the phenotypic and genotypic antimicrobial resistance identified on the E. coli isolated from postpartum cattle. Bacterial isolates (n = 148) were obtained from a larger cross-sectional study. Cows were categorized into one of three clinical groups before enrollment: metritis, cows with purulent discharge, and control cows. For genomic comparison, public genomes (n = 130) from cows with diarrhea, mastitis, and metritis were included in a genome-wide association study, to evaluate differences between the drug classes or the virulence factor category among clinical groups. A distinct E. coli genotype associated with metritis could not be identified. Instead, a high genetic diversity among the isolates from uterine sources was present. A virulence factor previously associated with metritis (fimH) using PCR was not associated with metritis. There was moderate accuracy for whole-genome sequencing to predict phenotypic resistance, which varied depending on the antimicrobial tested. Findings from this study contradict the traditional pathotype classification and the unique intrauterine E. coli genotype associated with metritis in dairy cows.IMPORTANCEMetritis is a common infectious disease in dairy cattle and the second most common reason for treating a cow with antimicrobials. The pathophysiology of the disease is complex and is not completely understood. Specific endometrial pathogenic Escherichia coli have been reported to be adapted to the endometrium and sometimes lead to uterine disease. Unfortunately, the specific genomic details of the endometrial-adapted isolates have not been investigated using enough genomes to represent the genomic diversity of this organism to identify specific virulence genes that are consistently associated with disease development and severity. Results from this study provide key microbial ecological advances by elucidating and challenging accepted concepts for the role of Intrauterine E. coli in metritis in dairy cattle, especially contradicting the existence of a unique intrauterine E. coli genotype associated with metritis in dairy cows, which was not found in our study.

Characterizing the gut phageome and phage-borne antimicrobial resistance genes in pigs

BackgroundMammalian intestine harbors a mass of phages that play important roles in maintaining gut microbial ecosystem and host health. Pig has become a common model for biomedical research and provides a large amount of meat for human consumption. However, the knowledge of gut phages in pigs is still limited.ResultsHere, we investigated the gut phageome in 112 pigs from seven pig breeds using PhaBOX strategy based on the metagenomic data. A total of 174,897 non-redundant gut phage genomes were assembled from 112 metagenomes. A total of 33,487 gut phage genomes were classified and these phages mainly belonged to phage families such as Ackermannviridae, Straboviridae, Peduoviridae, Zierdtviridae, Drexlerviridae, and Herelleviridae. The gut phages in seven pig breeds exhibited distinct communities and the gut phage communities changed with the age of pig. These gut phages were predicted to infect a broad range of 212 genera of prokaryotes, such as Candidatus Hamiltonella, Mycoplasma, Colwellia, and Lactobacillus. The data indicated that broad KEGG and CAZy functions were also enriched in gut phages of pigs. The gut phages also carried the antimicrobial resistance genes (ARGs) and the most abundant antimicrobial resistance genotype was diaminopyrimidine resistance.ConclusionsOur research delineates a landscape for gut phages in seven pig breeds and reveals that gut phages serve as a key reservoir of ARGs in pigs.53TvwP5ops6wwG1EthsGXZVideo

Utilizing co-abundances of antimicrobial resistance genes to identify potential co-selection in the resistome.

Understanding the collateral effects happening in a resistome can reveal previously unknown links between antimicrobial resistance genes (ARGs). Through the analysis of pairwise ARG abundances in 214K metagenomic samples, we observed that the co-abundance is highly dependent on the environmental context and argue that these correlations can be used to show the risk of co-selection occurring in different settings.

Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Multiplex microarray PCR Unyvero BCU system to accelerate relevant antimicrobial treatment in polymicrobial bloodstream infection.

To evaluate the performance of a rapid multiplex microarray-based method (Unyvero BCU system, BCU) to identify microorganisms and detect antimicrobial resistance directly from positive blood culture (BC) bottles with polymicrobial growth, and to assess relevance of information provided for timely guidance of polymicrobial bloodstream infection treatment. Accuracy, time-to-actionable results and potential impact of BCU on antimicrobial treatment were compared with those of standard of care during a prospective study for the sample analysis (November 2017-November 2018) and a retrospective study for the clinical data analysis and the time-to-result analysis. The study was complemented with an experimental study, based on spiked blood cultures to assess the ability of the method to detect antimicrobial resistance genes. Sixty-five clinical polymicrobial BC samples (163 total microorganisms) and 30 simulated polymicrobial BC samples (60 strains) were included. BCU reported 84.6% samples as polymicrobial, correctly identified all the bacteria of the mix for 72.3% samples (47/65) and detected bacteria that were missed by the conventional culture for 13.8% samples. All identifications and antimicrobial resistances were accurately detected for 61.5% (40/65) samples. Limitations concerned the detection of anaerobes, enterococci and enterobacterial susceptibility to third generation cephalosporins. BCU results would have guided antimicrobial treatment for 50.8% of the cases (33/65) in a timely and relevant manner, had no impact for 27.7% (18/65) and been misleading for 18.5% (12/65). Despite some limitations, the Unyvero BCU system is a rapid and reliable method for polymicrobial BC sample analysis.

Effect of environmental factors on conjugative transfer of antibiotic resistance genes in aquatic settings.

Antimicrobial-resistance genes (ARGs) are spread among bacteria by horizontal gene transfer, however, the effect of environmental factors on the dynamics of the ARG in water environments has not been very well understood. In this systematic review, we employed the regression tree algorithm to identify the environmental factors that facilitate/inhibit the transfer of ARGs via conjugation in planktonic/biofilm-formed bacterial cells based on the results of past relevant research. Escherichia coli strains were the most studied genus for conjugation experiments as donor/recipient in the intra-genera category. Conversely, Pseudomonas spp., Acinetobacter spp., and Salmonella spp. were studied primarily as recipients across inter-genera bacteria. The conjugation efficiency (ce) was found to be highly dependent on the incubation period. Some antibiotics, such as nitrofurantoin (at≥0.2µg ml-1) and kanamycin (at≥9.5mg l-1) as well as metallic compounds like mercury (II) chloride (HgCl2, ≥3µmol l-1), and vanadium (III) chloride (VCl3, ≥50µmol l-1) had enhancing effect on conjugation. The highest ce value (-0.90 log10) was achieved at 15°C-19°C, with linoleic acid concentrations<8mg l-1, a recognized conjugation inhibitor. Identifying critical environmental factors affecting ARG dissemination in aquatic environments will accelerate strategies to control their proliferation and combat antibiotic resistance.

β-lactamase expression induces collateral sensitivity in Escherichia coli

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, β-lactam resistance genes –encoding β-lactamases– stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the β-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile β-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that β-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of β-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

Insights into the genomic traits of Yersinia frederiksenii, Yersinia intermedia and Yersinia kristensenii isolated from diverse sources in Brazil.

Yersinia is an important genus comprising foodborne, zoonotic and pathogenic bacteria. On the other hand, species of the so-called group Yersinia enterocolitica-like are understudied and mostly characterized as non-pathogenic, despite of some reports of human infections. The present study aimed to provide genomic insights of Yersinia frederiksenii (YF), Yersinia intermedia (YI) and Yersinia kristensenii (YK) isolated worldwide. A total of 22 YF, 20 YI and 14 YK genomes were searched for antimicrobial resistance genes, plasmids, prophages, and virulence factors. Their phylogenomic relatedness was analyzed by Gegenees and core-genome multi-locus sequence typing. Beta-lactam resistance gene blaTEM-116 and five plasmids replicons (pYE854, ColRNAI, ColE10, Col(pHAD28) and IncN3) were detected in less than five genomes. A total of 59 prophages, 106 virulence markers of the Yersinia genus, associated to adherence, antiphagocytosis, exoenzymes, invasion, iron uptake, proteases, secretion systems and the O-antigen, and virulence factors associated to other 20 bacterial genera were detected. Phylogenomic analysis revealed high inter-species distinction and four highly diverse YF clusters. In conclusion, the results obtained through the analyses of YF, YI and YK genomes suggest the virulence potential of these strains due to the broad diversity and high frequency of prophages and virulence factors found. Phylogenetic analyses were able to correctly distinguish these closely related species and show the presence of different genetic subgroups. These data contributed for a better understanding of YF, YI and YK virulence-associated features and global genetic diversity, and reinforced the need for better characterization of these Y. enterocolitica-like species considered non-pathogenic.

β-lactamase and macrolide resistance gene carriage in E. coli isolates among children discharged from inpatient care in western Kenya: a cross-sectional study

Abstract Background Antimicrobial resistance (AMR) is a global threat to infectious disease control, particularly among recently hospitalized children. We sought to determine the prevalence and mitigating factors of resistance in enteric Escherichia coli among children discharged from health facilities in western Kenya. Methods Between June 2016 and November 2019, children aged 1-59 months were enrolled at the point of discharge from hospital. E. coli was isolated by microbiological culture from rectal swabs at baseline. β-lactamases and macrolide resistance-conferring genes were detected by PCR. The modified Poisson regression model was used to assess the predictors mph(A) and CTX-M-type ESBL. Results Of the 238 children whose E. coli isolates were tested, 91 (38.2%) and 109 (45.8%) had detectable CTX-M-type ESBL and mph(A) genes, respectively. Antibiotic treatment during hospitalization (adjusted prevalence ratio [aPR]=2.47; 95%CI:1.12-5.43, p=0.025), length of hospitalization (aPR=1.42; 95%CI:1.00-2.01, p=0.052), and the practice of open defecation (aPR=2.47; 95%CI:1.40-4.36, p=0.002) were independent predictors for CTX-M-type ESBL and for mph(A) genes. Pneumococcal vaccination was associated with a 43% lower likelihood of CTX-M-type ESBL (aPR=0.57; 95%CI:0.38-0.85, p=0.005), whilst measles vaccination was associated with a 32% lower likelihood of mph(A) gene (aPR=0.68; 95%CI:0.49-0.93, p=0.017) in E. coli isolates. Conclusions Among children discharged from hospital, history of vaccination, shorter hospital stay, lack of in-hospital antibiotic exposure and improved sanitation were associated with lower likelihood of AMR genes. AMR control programs should consider strategies beyond antimicrobial stewardship, including improvements in sanitation, increased vaccine coverage and the development of novel vaccines, to mitigate the continued spread of AMR.

The Molecular Mouse System: A New Useful Tool for Guiding Antimicrobial Therapy in Critically Ill Septic Patients.

Bloodstream infections (BSI) caused by multidrug-resistant (MDR) bacteria, pose a major threat for patients, especially for those who are immunosuppressed. Rapid pathogen detection and characterization from positive blood cultures are crucial in the management of patients with BSI to enable an adequate and timely antimicrobial therapy. This study aimed to investigate the potential role of the Molecular Mouse system, a new CE IVD molecular test designed to rapidly detect the causative agents of bacteremia and their resistance determinants, in the management of the therapy in critically ill patients. Agreement between the results of the Molecular Mouse and the conventional routine method was also considered. Overall, 100 positive blood cultures were collected from septic critically ill patients from May 2023 to January 2024 and analyzed with Molecular Mouse and routine protocols. The new instrument consistently agreed with the routine protocols in the case of monomicrobial blood cultures, while some discrepancies were obtained in the polymicrobial samples. Antimicrobial resistance genes were detected in 35 samples, with vanA and CTX-M-1/9 groups being the most frequently detected targets. Therapy was adjusted in 42 critically ill patients confirming the importance of new rapid molecular tests in the management of positive blood cultures, to adjust empirical therapy and use new antibiotics accurately.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively.DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Antimicrobial resistance, enterotoxin and biofilm production genes in Staphylococcus spp. isolated from facilities and fomites in veterinary hospital in the Caatinga biome.

The Caatinga biome occurs only in Brazil and offers epidemiological conditions that should be assessed differently from other regions of Brazil and the world. Thus, the aim of this survey was to identify antimicrobial resistance, enterotoxin and biofilm production genes in Staphylococcus spp. isolated from facilities and fomites in a veterinary hospital in Caatinga biome. Samples were collected from surfaces of small animal clinical care tables (n =8), cages in the dog and cat hospitalisation sector and animals with infectious diseases (n = 21), small animal surgical centre (n =8), sterilisation sector (n =7) and stethoscopes (n = 32) by using sterile swabs. Bacterial isolation and identification, antimicrobial resistance phenotypic test and molecular detection of antimicrobial resistance, biofilm formation and enterotoxin genes were carried out. Ninety-five bacterial isolates were obtained, and 29 (30.5%) were identified as Staphylococcus spp. Overall, 13 isolates (44.8%) of six species of Staphylococcus spp. showed antimicrobial resistance profile, as well as S. haemolyticus expressed phenotypic profile of multidrug resistance. The antimicrobials with the highest resistance rates were penicillin and tetracycline. The most frequent resistance genes were blaZ and tetM, both detected in 10 (76.9%) isolates. The mecA, tetL and tetK genes had frequencies of 38.5% (5/13), 23.1% (3/13) and 15.4% (2/13), respectively. The biofilm production marker, icaD gene, was detected in one S. sciuri strain. SEE gene, which encodes enterotoxins, was detected in 15.4% (2/13) of the strains (S. pseudintermedius and S. intermedius). The occurrence of Staphylococcus spp. carrying resistance genes to diferent classes of antimicrobials, presenting MDR phenotypic pattern and carrying enterotoxins and biofim encoding genes recovered from veterinary hospital facilities and fomites in the Caatinga biome reinforce the need to implement prevention cares in veterinary practices to avoid One Health-concerning conditions.

The mobilome of Staphylococcus aureus from wild ungulates reveals epidemiological links at the animal-human interface

Staphylococcus aureus thrives at animal-human-environment interfaces. A large-scale work from our group indicated that antimicrobial resistance (AMR) in commensal S. aureus strains from wild ungulates is associated with agricultural land cover and livestock farming, raising the hypothesis that AMR genes in wildlife strains may originate from different hosts, namely via exchange of mobile genetic elements (MGE). In this work, we generate the largest available dataset of S. aureus draft genomes from wild ungulates in Portugal and explore their mobilome, which can determine important traits such as AMR, virulence, and host specificity, to understand MGE exchange. Core genome multi-locus sequence typing based on 98 newly generated draft genomes and 101 publicly available genomes from Portugal demonstrated that the genomic relatedness of S. aureus from wild ungulates assigned to livestock-associated sequence types (ST) is greater compared to wild ungulate isolates assigned to human-associated STs. Screening of host specificity determinants disclosed the unexpected presence in wildlife of the immune evasion cluster encoded in φSa3 prophage, described as a human-specific virulence determinant. Additionally, two plasmids, pAVX and pETB, previously associated with avian species and humans, respectively, and the Tn553 transposon were detected. Both pETB and Tn553 encode penicillin resistance through blaZ. Pangenome analysis of wild ungulate isolates shows a core genome fraction of 2133 genes, with isolates assigned to ST72 and ST3224 being distinguished from the remaining by MGEs, although there is no reported role of these in adaptation to wildlife. AMR related gene clusters found in the shell genome are directly linked to resistance against penicillin, macrolides, fosfomycin, and aminoglycosides, and they represent mobile ARGs. Altogether, our findings support epidemiological interactions of human and non-human hosts at interfaces, with MGE exchange, including AMR determinants, associated with putative indirect movements of S. aureus among human and wildlife hosts that might be bridged by livestock.

River waters in Greece: A reservoir for clinically relevant extended-spectrum-β-lactamases-producing Escherichia coli

In the current study, the genotypic characteristics such as antimicrobial resistance and virulence genes, and plasmid replicons and phenotypic characteristics such as biofilm formation and antimicrobial resistance of 87 extended-spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-Ec) isolated from 7 water bodies in northern Greece were investigated. Our data show a high prevalence (60.0 %) of ESBL-Ec in surface waters that exhibit high genetic diversity, suggesting multiple sources of their transmission into the aquatic environment. When evaluating the antimicrobial resistance of isolates, wide variation in their resistance profiles has been detected, with all isolates being multi-drug resistant (MDR). Regarding biofilm formation capacity and phylogenetic groups, the majority (54.0 %, 47/87) of ESBL-Ec were classified as no biofilm producers mainly assigned to phylogroup A (35.6 %; 31/87), followed by B2 (26.5 %; 23/87). PCR screening showed that a high proportion of the isolates tested positive for the blaCTX-M-1 group genes (69 %, 60/87), followed by blaTEM (55.2 %, 48/87), blaOXA (25.3 %, 22/87) and blaCTX-M-9 (17.2 %, 15/87). A subset of 28 ESBL-Ec strains was further investigated by applying whole genome sequencing (WGS), and among them, certain clinically significant sequence types were identified, such as ST131 and ST10. The corresponding in silico analysis predicted all these isolates as human pathogens, while a significant proportion of WGS-ESBL-Ec were assigned to extraintestinal pathogenic E. coli (ExPEC; 32.1 %), and urinary pathogenic E. coli (UPEC; 28.6 %) pathotypes. Comparative phylogenetic analysis, showed that the genomes of the ST131-O25:H4-H30 isolates are genetically linked to the human clinical strains. Here, we report for the first time the detection of a plasmid-mediated mobile colistin resistance gene in ESBL-Ec in Greece isolated from an environmental source. Overall, this study underlines the role of surface waters as a reservoir for antibiotic resistance genes and for presumptive pathogenic ESBL-Ec.

Phenomics and genomic features of Enterococcus avium IRMC1622a isolated from a clinical sample of hospitalized patient

BackgroundEnterococcus avium (E. avium) is a Gram-positive nosocomial pathogen that is commonly isolated from the alimentary tract. The objective of this functional genomics study was to identify the resistant genes by analyzing the genome of E. avium IRMC1622a, a type of bacteria found in feces collected from a patient at a Saudi Arabian tertiary hospital. MethodsThe bacterial strain IRMC1622a was identified by 16 S rRNA sequencing as Enterococcus sp. The resistance phenomics were performed using VITEK® 2, and morphological analysis was achieved using a scanning electron microscope (SEM). Finally, the whole bacterial genome of the bacterial strain IRMC1622a was subjected to sequencing during October 2023 using Oxford Nanopore long-read sequencing technology, and mining for resistant genes. ResultsThe results of antimicrobial resistant phenomics indicated that the IRMC1622a strain was sensitive to all tested antimicrobial agents except for erythromycin, and the same result was confirmed by genomic analysis in addition to other classes of antibiotics. SEM showed E. avium IRMC1622a is ovoid shape, in single cells (L 1.2797 ± 0.1490 µm), in pairs (L 1.7333 ± 0.1054 µm), and in chains (L 2.44033 ± 0.1978 µm). The E. avium IRMC1622a genome has 14 (in CARD) antimicrobial resistance genes that were identified with several mechanisms of antimicrobial resistance, such as the efflux pump and conferring antibiotic resistance. The present study revealed that the E. avium IRMC1622a genome contains a high number of genes associated with virulence factors, and 14 matched pathogenic protein families and predicted as human pathogen (probability score 0.855). We report two (ISEnfa4 and ISEfa5) mobile genetic elements for the first time in the E. avium genome. ConclusionsThe study concludes that E. avium IRMC1622a is susceptible to all tested antibacterials except erythromycin. The IRMC1622a has 14 genes encoding antimicrobial resistance mechanisms, including the efflux pump and conferring antibiotic resistance. This could indicate a potential rise in E. avium resistance in healthcare facilities. These observations may raise concerns regarding E. avium resistance in healthcare. We need more research to understand the pathophysiology of E. avium, which leads to hospital-acquired infections.

Clinical Utility of Blood Culture Identification 2 Panel in Flagged Blood Culture Samples from the Intensive Care Unit of a Tertiary Care Hospital.

The availability of rapid diagnostic platforms for positive blood cultures has accelerated the speed at which the clinical microbiology laboratory can identify the causative organism and facilitate early appropriate antimicrobial therapy. There is a paucity of data regarding the clinical utility of the blood culture identification 2 (BCID2) panel test and its correlation with phenotypic drug susceptibility testing (DST) in flagged blood culture bottles from intensive care units (ICUs) in countries such as India, which have high rates of multidrug-resistant gram-negative bacteria (MDR-GNB). We conducted a retrospective observational study in a tertiary care ICU on 200 patients above 18 years of age in whom a BCID2 test was ordered when blood cultures flagged positive. We found 99% concordance between BCID2 and cultures in the identification of bacteria and yeasts and 96.5% concordance between phenotypic and genotypic DST. Furthermore, BCID2 was available about 1.5 days earlier than conventional ID and DST and played a key role in tailoring antimicrobials in 82.5% of the patients. Polymyxin-based therapy was discontinued earlier after an empiric dose in 138 patients (69%) based on BCID2 reports. In critically ill patients with monomicrobial bacteremia, BCID2 rapidly identifies bacteria and antimicrobial resistance (AMR) genes and is significantly faster than conventional culture and sensitivity testing. Antibiotics were escalated in more than a third of patients and de-escalated in almost a fifth on the same day. We recommend that all ICUs routinely incorporate the test in their antibiotic decision-making process and in antimicrobial stewardship. Vineeth VK, Nambi PS, Gopalakrishnan R, Sethuraman N, Ramanathan Y, Chandran C, et al. Clinical Utility of Blood Culture Identification 2 Panel in Flagged Blood Culture Samples from the Intensive Care Unit of a Tertiary Care Hospital. Indian J Crit Care Med 2024;28(5):461-466.

Co-occurrence of the cephalosporinase cepA and carbapenemase cfiA genes in a Bacteroides fragilis division II strain, an unexpected finding.

Bacteroides fragilis, an anaerobic gut bacterium and opportunistic pathogen, comprises two genetically divergent groups (or divisions) at the species level. Differences exist both in the core and accessory genomes and the beta-lactamase genes, with the cephalosporinase gene cepA represented only in division I and the carbapenemase gene cfiA only in division II. Multidrug resistance in a clinical B. fragilis strain was examined by whole-genome sequencing. Strain CNM20200260 carried the antimicrobial resistance genes cepA, cfiA2, ant(6'), erm(F), mef(En2), est(T), tet(Q) and cat(A), along with 82-Phe mutation in gyrA (together with 47 amino acid changes in gyrA/B and parC/parE). bexA/B and other efflux pump genes were also observed. None of the detected insertion sequences was located upstream of cfiA2. The genome-based taxonomy coefficients (average nucleotide identity, DNA-DNA hybridization similarity and difference in genomic G + C%) with respect to genomes of the strains of B. fragilis division II and the novel species Bacteroides hominis (both cfiA-positive) met the criteria for CNM20200260 to belong to either species (>95%, >70% and <1%, respectively). No such similarity was seen with type strain NCTC 9343 or the representative genome FDAARGOS 1225 of B. fragilis (division I, cfiA-negative). Strain CNM20200260 harboured four out of nine Kyoto Encyclopedia of Genes and Genomes orthologues defined for division I and one of two defined for division II. This is the first description of the co-occurrence of cepA and cfiA in a Bacteroides strain, confirming the complexity of the taxonomy of this species.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377bp and 3,285,803bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

Impact of Sulfadoxine-Pyrimethamine and Dihydroartemisinin-Piperaquine as Intermittent Preventive Treatment in Pregnancy on Stool Antimicrobial Resistance Gene Abundance.

Increasing antimicrobial resistance (AMR) is a global public health emergency. Although chemoprevention has improved malaria-related pregnancy outcomes, the downstream effects on AMR have not been characterized. We compared the abundance of 10 AMR genes in stool samples from pregnant women receiving sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment against malaria in pregnancy (IPTp) to that in samples from women receiving dihydroartemisinin-piperaquine (DP) for IPTp. All participants had at least one AMR gene at baseline. Mean quantities of the antifolate gene dfrA17 were increased after two or more doses of SP (mean difference = 1.6, 95% CI: 0.4-2.7, P = 0.008). Antimicrobial resistance gene abundance tended to increase from baseline in SP recipients compared with a downward trend in the DP group. Overall, IPTp-SP had minimal effects on the abundance of antifolate resistance genes (except for dfrA17), potentially owing to a high starting prevalence. However, the trend toward increasing AMR in SP recipients warrants further studies.