Phenotypic Antimicrobial Susceptibility Research Articles

Epidemiology, Antimicrobial Resistance, and Virulence Determinants of Group B Streptococcus in an Australian Setting.

Streptococcus agalactiae [group B Streptococcus (GBS)] is a major neonatal pathogen and also causes invasive disease in non-pregnant adults. One hundred GBS isolates (n = 50 invasive disease and n = 50 colonizing pregnant women) were characterized using capsular serotyping by latex agglutination, antimicrobial susceptibility testing, and whole genome sequencing (WGS). All isolates were susceptible to penicillin, 32% were resistant to clindamycin. Of these, two isolates had reduced susceptibility to ceftriaxone (MIC 0.75 mg/L) and were found to have unique alleles at pbp2X and pbp1A. Capsular serotypes Ia (18%), III (18%), Ib (14%), V (12%), and VI (11%) were most common and comparison of latex agglutination and capsular genotyping by WGS showed 71% agreement. Less common capsular genotypes VI–VIII represented 15% of isolates, indicating that a significant proportion may not be targeted by the proposed pentavalent or hexavalent vaccines under development. WGS is a useful aid in GBS surveillance and shows correlation to phenotypic serotyping and antimicrobial susceptibility data.

One Day in Denmark: Comparison of Phenotypic and Genotypic Antimicrobial Susceptibility Testing in Bacterial Isolates From Clinical Settings.

Antimicrobial susceptibility testing (AST) should be fast and accurate, leading to proper interventions and therapeutic success. Clinical microbiology laboratories rely on phenotypic methods, but the continuous improvement and decrease in the cost of whole-genome sequencing (WGS) technologies make them an attractive alternative. Studies evaluating the performance of WGS-based prediction of antimicrobial resistance (AMR) for selected bacterial species have shown promising results. There are, however, significant gaps in the literature evaluating the applicability of WGS as a diagnostics method in real-life clinical settings against the range of bacterial pathogens experienced there. Thus, we compared standard phenotypic AST results with WGS-based predictions of AMR profiles in bacterial isolates without preselection of defined species, to evaluate the applicability of WGS as a diagnostics method in clinical settings. We collected all bacterial isolates processed by all Danish Clinical Microbiology Laboratories in 1 day. We randomly selected 500 isolates without any preselection of species. We performed AST through standard broth microdilution (BMD) for 488 isolates (n = 6,487 phenotypic AST results) and compared results with in silico antibiograms obtained through WGS (Illumina NextSeq) followed by bioinformatics analyses using ResFinder 4.0 (n = 5,229 comparisons). A higher proportion of AMR was observed for Gram-negative bacteria (10.9%) than for Gram-positive bacteria (6.1%). Comparison of BMD with WGS data yielded a concordance of 91.7%, with discordant results mainly due to phenotypically susceptible isolates harboring genetic AMR determinants. These cases correspond to 6.2% of all isolate-antimicrobial combinations analyzed and to 6.8% of all phenotypically susceptible combinations. We detected fewer cases of phenotypically resistant isolates without any known genetic resistance mechanism, particularly 2.1% of all combinations analyzed, which corresponded to 26.4% of all detected phenotypic resistances. Most discordances were observed for specific combinations of species-antimicrobial: macrolides and tetracycline in streptococci, ciprofloxacin and β-lactams in combination with β-lactamase inhibitors in Enterobacterales, and most antimicrobials in Pseudomonas aeruginosa. WGS has the potential to be used for surveillance and routine clinical microbiology. However, in clinical microbiology settings and especially for certain species and antimicrobial agent combinations, further developments in AMR gene databases are needed to ensure higher concordance between in silico predictions and expected phenotypic AMR profiles.

Metagenomic DNA sequencing for semi-quantitative pathogen detection from urine: a prospective, laboratory-based, proof-of-concept study

Semi-quantitative bacterial culture is the reference standard to diagnose urinary tract infection, but culture is time-consuming and can be unreliable if patients are receiving antibiotics. Metagenomics could increase diagnostic accuracy and speed by sequencing the microbiota and resistome directly from urine. We aimed to compare metagenomics to culture for semi-quantitative pathogen and resistome detection from urine. In this proof-of-concept study, we prospectively included consecutive urine samples from a clinical diagnostic laboratory in Amsterdam. Urine samples were screened by DNA concentration, followed by PCR-free metagenomic sequencing of randomly selected samples with a high concentration of DNA (culture positive and negative). A diagnostic index was calculated as the product of DNA concentration and fraction of pathogen reads. We compared results with semi-quantitative culture using area under the receiver operating characteristic curve (AUROC) analyses. We used ResFinder and PointFinder for resistance gene detection and compared results to phenotypic antimicrobial susceptibility testing for six antibiotics commonly used for urinary tract infection treatment: nitrofurantoin, ciprofloxacin, fosfomycin, cotrimoxazole, ceftazidime, and ceftriaxone. We screened 529 urine samples of which 86 were sequenced (43 culture positive and 43 culture negative). The AUROC of the DNA concentration-based screening was 0·85 (95% CI 0·81-0·89). At a cutoff value of 6·0 ng/mL, culture positivity was ruled out with a negative predictive value of 91% (95% CI 87-93; 26 of 297 samples), reducing the number of samples requiring sequencing by 56% (297 of 529 samples). The AUROC of the diagnostic index was 0·87 (95% CI 0·79-0·95). A diagnostic index cutoff value of 17·2 yielded a positive predictive value of 93% (95% CI 85-97) and a negative predictive value of 69% (55-80), correcting for a culture-positive prevalence of 66%. Gram-positive pathogens explained eight (89%) of the nine false-negative metagenomic test results. Agreement of phenotypic and genotypic antimicrobial susceptibility testing varied between 71% (22 of 31 samples) and 100% (six of six samples), depending on the antibiotic tested. This study provides proof-of-concept of metagenomic semi-quantitative pathogen and resistome detection for the diagnosis of urinary tract infection. The findings warrant prospective clinical validation of the value of this approach in informing patient management and care. EU Horizon 2020 Research and Innovation Programme.

Comparison of genome-derived and phenotypic antimicrobial resistance profiles of Shigella species isolated from patients with symptoms of gastrointestinal disease in England, 2015-2020

Increasing antimicrobial resistance (AMR) in Shigellaspecies is a global public health problem. We compared genotypic and phenotypic methods for the detection of AMR in Shigellaspecies to evaluate the use of genome data for surveillance and monitoring of emerging AMR. Whole genome sequencing (WGS) data from 388 isolates of all four Shigella species were analysed for the presence/absence of specific AMR determinants and selected accordingly. Phenotypic antimicrobial susceptibility testing was performed using in-agar dilution on all viable and pure isolates (n=358). The genotypic and phenotypic AMR profiles were then compared. There were 335 (93.6%) isolates resistant to at least one antimicrobial and 222 (62%) isolates were multi-drug resistant, of which the majority (77%) were associated with foreign travel. Out of a possible 2864 isolate/antimicrobial class combinations, we identified 119 unexpected results, giving an overall concordance of 96.8% between the two methods. There were 54 samples that had an AMR determinant expected to confer resistance that were phenotypically susceptible, of which 31/54 (57.4%) were associated with tetracycline resistance and trimethoprim-sulfamethoxazole resistance. There were 65 that were phenotypically resistant to a specific antimicrobial class, but no AMR determinant was detected, of which 32/65 (49.2%) were associated with isolates harbouring a single gyrA mutation and exhibiting an unexpectedly high minimum inhibitory concentration (MIC) to ciprofloxacin. Although comparisons between both methods showed good correlation between the genotypic and phenotypic AMR profiles, phenotypic monitoring is required to identify novel AMR mechanisms and to update reference database used for WGS analysis.

Prevalence and antimicrobial resistance of Salmonella in conventional and no antibiotics ever broiler farms in the United States

Non-typhoidal Salmonella is the second most common bacterial cause of foodborne illness in the United States. More than 50% of the poultry meat sold in the U.S. is categorized as no antibiotics ever (NAE), yet it is unclear how these practices affect the prevalence and antimicrobial resistance (AMR) of Salmonella . The aim of this study was to evaluate and compare the prevalence of Salmonella and the incidence of AMR in conventional (n=2) and NAE (n = 2) broiler farms in the Southeast US. Broiler cecal (n = 420) and ileal contents (n = 420), along with litter (n = 24), feed (n = 24) and water (n = 24) samples were collected from four broiler farms. Sample collection was carried out during a full production cycle and following the birds to the processing facilities. The BAX® Real-Time PCR system was used to detect Salmonella positive samples. Phenotypic antimicrobial susceptibility was evaluated following the National Antimicrobial Resistance Monitoring System (NARMS) method, and whole genome sequencing (WGS) was carried out for screening of AMR genes. The software tool SeqSero2 and the McOutbryk SNP calling pipeline were used for serotype identification and phylogenetic analysis, respectively. Conventional broilers had the highest Salmonella prevalence (80% and 73%; P ≤ 0.05) at day 21. The Salmonella prevalence declined as the birds aged. Antimicrobial resistance among Salmonella isolates from all sample types was high in all four farms (76%), exhibiting frequent resistance to tetracycline (76%) and streptomycin (70%). Salmonella isolates recovered from NAE broilers carried higher (P ≤ 0.05) rates of AMR than isolates from conventionally raised chicken. Salmonella enterica serotype Kentucky was the predominant serotype, harboring genes tet(B), aph(3″)-Ib and aph(6)-Id (which encode resistance to tetracycline and streptomycin, respectively). Farm-related strains of the serotype were identified, exhibiting persistence in the litter environment. • Broilers from NAE farms had lower Salmonella prevalence in the Southeast US. • Resistance to tetracycline and streptomycin among Salmonella was widespread. • Salmonella Kentucky was the predominant serotype on farms in the Southeast US. • Phylogeny of S. Kentucky strains exhibited farm-specific association. • Antimicrobial resistant genes tet(B), aph(3″)‐Ib , aph(6)‐Id and fosA7 were found.

Environmental surveillance of ESBL and carbapenemase-producing gram-negative bacteria in a Ghanaian Tertiary Hospital

BackgroundThe burden of antibiotic resistant infection is mainly felt in low-to-middle income countries, where the rate of antimicrobial resistance is largely under-surveyed and under huge pressure from unregulated, disparate and often self-guided access to antimicrobials. Nosocomial infections from hospital environments have been shown to be a particularly prevalent source of multi-drug resistant strains, yet surveillance of hospital environmental contamination is often not investigated.MethodsThe study was prospective, observational and cross-sectional, sampling 231 high and low touch surfaces from 15th March to 13th April 2021, from five wards in the Cape Coast Teaching Hospital, Ghana. Microbial growth in the presence of vancomycin and either meropenem or cefotaxime was examined and bacterial species were identified by MALDI-TOF. The presence of common extended-spectrum β-lactamases (ESBL) and carbapenemase antimicrobial resistance genes (ARG) were identified through PCR screening, which were confirmed by phenotypic antimicrobial susceptibility determination. Isolates positive for carbapenem resistance genes were sequenced using a multi-platform approach.ResultsWe recovered microbial growth from 99% of swabs (n = 229/231) plated on agar in the absence of antimicrobials. Multiple sites were found to be colonised with resistant bacteria throughout the hospital setting. Bacteria with multi-drug resistance and ARG of concern were isolated from high and low touch points with evidence of strain dissemination throughout the environment. A total of 21 differing species of bacteria carrying ARG were isolated. The high prevalence of Acinetobacter baumannii carrying blaNDM-1 observed was further characterised by whole genome sequencing and phylogenetic analysis to determine the relationship between resistant strains found in different wards.ConclusionEvidence of multiple clonal incursions of MDR bacteria of high sepsis risk were found in two separate wards for a regional hospital in Ghana. The prevalence of multiple blaNDM carrying species in combination with combinations of ESBLs was particularly concerning and unexpected in Africa. We also identify strains carrying tet(X3), blaVIM-5 or blaDIM-1 showing a high diversity of carbapenamases present as a reservoir in a hospital setting. Findings of multi-drug resistant bacteria from multiple environmental sites throughout the hospital will inform future IPC practices and aid research prioritisation for AMR in Ghana.

Genetic Diversity, Distribution, and Genomic Characterization of Antibiotic Resistance and Virulence of Clinical Pseudomonas aeruginosa Strains in Kenya.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide. It can produce a range of debilitating infections, have a propensity for developing antimicrobial resistance, and present with a variety of potent virulence factors. This study investigated the sequence types (ST), phenotypic antimicrobial susceptibility profiles, and resistance and virulence genes among clinical isolates from urinary tract and skin and soft tissue infections. Fifty-six P. aeruginosa clinical isolates were obtained from six medical centers across five counties in Kenya between 2015 and 2020. Whole-genome sequencing (WGS) was performed to conduct genomic characterization, sequence typing, and phylogenetic analysis of the isolates. Results showed the presence of globally distributed high-risk clones (ST244 and ST357), local high-risk clones (ST2025, ST455, and ST233), and a novel multidrug-resistant (MDR) clone carrying virulence genes (ST3674). Furthermore, 31% of the study isolates were found to be MDR with phenotypic resistance to a variety of antibiotics, including piperacillin (79%), ticarcillin-clavulanic acid (57%), meropenem (34%), levofloxacin (70%), and cefepime (32%). Several resistance genes were identified, including carbapenemases VIM-6 (ST1203) and NDM-1 (ST357), fluoroquinolone genes, crpP, and qnrVCi, while 14 and 22 different chromosomal mutations were detected in the gyrA and parC genes, respectively. All isolates contained at least three virulence genes. Among the virulence genes identified, phzB1 was the most abundant (50/56, 89%). About 21% (12/56) of the isolates had the exoU+/exoS- genotype, while 73% (41/56) of the isolates had the exoS+/exoU- genotype. This study also discovered 12 novel lineages of P. aeruginosa, of which one (ST3674) demonstrated both extensive antimicrobial resistance and the highest number of virulence genes (236/242, 98%). Although most high-risk clones were detected in Nairobi County, high-risk and clones of interest were found throughout the country, indicating the local spread of global epidemic clones and the emergence of new strains. Thus, this study illustrates the urgent need for coordinated local, regional, and international antimicrobial resistance surveillance efforts.

Bacterial genotypic and patient risk factors for adverse outcomes in Escherichia coli bloodstream infections: a prospective molecular epidemiological study.

Objectives Escherichia coli bloodstream infections have shown a sustained increase in England, for reasons that are unknown. Furthermore, the contribution of MDR lineages such as ST131 to overall E. coli disease burden and outcome is undetermined.MethodsWe genome-sequenced E. coli blood isolates from all patients with E. coli bacteraemia in north-west London from July 2015 to August 2016 and assigned MLST genotypes, virulence factors and AMR genes to all isolates. Isolate STs were then linked to phenotypic antimicrobial susceptibility, patient demographics and clinical outcome data to explore relationships between the E. coli STs, patient factors and outcomes.ResultsA total of 551 E. coli genomes were analysed. Four STs (ST131, 21.2%; ST73, 14.5%; ST69, 9.3%; and ST95, 8.2%) accounted for over half of cases. E. coli genotype ST131-C2 was associated with phenotypic non-susceptibility to quinolones, third-generation cephalosporins, amoxicillin, amoxicillin/clavulanic acid, gentamicin and trimethoprim. Among 300 patients from whom outcome was known, an association between the ST131-C2 lineage and longer length of stay was detected, although multivariable regression modelling did not demonstrate an association between E. coli ST and mortality. Several unexpected associations were identified between gentamicin non-susceptibility, ethnicity, sex and adverse outcomes, requiring further research.ConclusionsAlthough E. coli ST was associated with defined antimicrobial non-susceptibility patterns and prolonged length of stay, E. coli ST was not associated with increased mortality. ST131 has outcompeted other lineages in north-west London. Where ST131 is prevalent, caution is required when devising empiric regimens for suspected Gram-negative sepsis, in particular the pairing of β-lactam agents with gentamicin.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

ObjectivesTo develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug.MethodsThe Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid.ResultsWe observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes.ConclusionsIn light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Phytochemical screening, toxicity and antimicrobial activity of different Mimosa tenuiflora extracts on Aeromonas strains

The indiscriminate use of synthetic veterinary drugs in fish farms for disease control has caused recurring environmental pollution and reduced productivity; however, the search for ecologically viable alternatives is increasing. Thus, this study aimed to evaluate the phytochemical characterization of the hexanic, methanolic, and aqueous extracts of black jurema (M. tenuiflora), and their antimicrobial activity against strains of Aeromonas, and acute toxicity (LC50) to fingerlings of O. niloticus. The isolates were identified, and phenotypic virulence and antimicrobial susceptibility tests were performed. A. caviae, and A. veronii bv. veronii showed 75.0 - 87.5% positivity for the virulence factors tested, and resistance to the antimicrobials ampicillin, amoxicillin, erythromycin, and tetracycline of 67.0% and 50.0%, respectively. Phytochemical screening of black jurema extracts detected phenols, flavonoids, tannins, saponins, alkaloids, and steroids/triterpenoids, with methanol proving to be more efficient in the extraction of metabolites. The methanolic and aqueous extracts showed moderate antimicrobial activity, with minimum inhibitory concentration and minimum bactericidal concentration of 250 μg mL-1, and the methanolic extract revealed an LC50 of 40 μg mL-1 for O. niloticus. This study demonstrated the efficiency of the in vitro antimicrobial activity of M. tenuiflora extracts, and their use in vivo in the treatment or prophylaxis in fish farming can be investigated to replace the use of synthetic antimicrobials.

Need for standardization and harmonization of Helicobacter pylori antimicrobial susceptibility testing.

As with other infectious diseases, Helicobacter pylori eradication regimens should be guided by susceptibility testing to achieve excellent success rate, especially in the era of high antibiotic resistance. However, susceptibility testing for H.pylori is rarely performed, which can be partly ascribed to the current lack of standardization of testing methods and the lack of unified consensus on the antibiotic resistance breakpoints. The aim of this review was to call for an international consensus on standardization and harmonization of H.pylori susceptibility testing. We summarize and compare the advantages and disadvantages of four different phenotypic antimicrobial susceptibility testing (AST) methods (agar dilution, E-test, disk diffusion, and broth microdilution) and the molecular susceptibility testing method for H.pylori. The standard phenotypic testing methods and the molecular testing methods have their own advantages and disadvantages. Compared to the standard phenotypic methods, the molecular testing method does not require successful H.pylori culture, and therefore, is much more rapid and convenient for clinical use. However, the currently available molecular testing method is only suitable for detecting clarithromycin and quinolone susceptibility profiles in H.pylori. Although the standard AST is time-consuming, it is currently the only way to test the susceptibility of H.pylori to all the commonly used antibiotics. To make H.pylori susceptibility testing become a clinical routine, an international consensus on standardization and harmonization of H.pylori AST is needed. Future efforts are needed for optimizing broth culture of H.pylori, and developing commercial AST plates for achieving high throughput and automated susceptibility testing for H.pylori.

Correlation between Phenotypic and In Silico Detection of Antimicrobial Resistance in Salmonella enterica in Canada Using Staramr.

Whole genome sequencing (WGS) of Salmonella supports both molecular typing and detection of antimicrobial resistance (AMR). Here, we evaluated the correlation between phenotypic antimicrobial susceptibility testing (AST) and in silico prediction of AMR from WGS in Salmonella enterica (n = 1321) isolated from human infections in Canada. Phenotypic AMR results from broth microdilution testing were used as the gold standard. To facilitate high-throughput prediction of AMR from genome assemblies, we created a tool called Staramr, which incorporates the ResFinder and PointFinder databases and a custom gene-drug key for antibiogram prediction. Overall, there was 99% concordance between phenotypic and genotypic detection of categorical resistance for 14 antimicrobials in 1321 isolates (18,305 of 18,494 results in agreement). We observed an average sensitivity of 91.2% (range 80.5–100%), a specificity of 99.7% (98.6–100%), a positive predictive value of 95.4% (68.2–100%), and a negative predictive value of 99.1% (95.6–100%). The positive predictive value of gentamicin was 68%, due to seven isolates that carried aac(3)-IVa, which conferred MICs just below the breakpoint of resistance. Genetic mechanisms of resistance in these 1321 isolates included 64 unique acquired alleles and mutations in three chromosomal genes. In general, in silico prediction of AMR in Salmonella was reliable compared to the gold standard of broth microdilution. WGS can provide higher-resolution data on the epidemiology of resistance mechanisms and the emergence of new resistance alleles.

Combinatorial screening SlipChip for rapid phenotypic antimicrobial susceptibility testing.

Antimicrobial resistance (AMR) by bacteria is a serious global threat, and a rapid, high-throughput, and easy-to-use phenotypic antimicrobial susceptibility testing (AST) method is essential for making timely treatment decisions and controlling the spread of antibiotic resistant micro-organisms. Traditional culture-based methods are time-consuming, and their capability to screen against a large number of different conditions is limited; meanwhile genotypic based methods, including sequencing and PCR based methods, are constrained by rarely identified resistance genes and complicated resistance mechanisms. Here, a combinatorial-screening SlipChip (cs-SlipChip) containing 192 nanoliter-sized compartments is developed which can perform high-throughput phenotypic AST within three hours by monitoring the bacterial growth within nanoliter-sized droplets with bright-field imaging and analyzing the changes in bacterial number and morphology. The minimum inhibitory concentration (MIC) of Escherichia coli ATCC 25922 against four antibiotics (ampicillin, ciprofloxacin, ceftazidime, and nitrofurantoin) can be measured in one chip within 3 hours. Furthermore, five antibiotic-resistant E. coli strains were isolated from patients diagnosed with urinary tract infections (UTIs), and an individual isolate was tested using four antibiotics and eleven antibiotic combinations simultaneously with three different concentrations of each. The results from the cs-SlipChip agree with those of a VITEK 2 automated system. This cs-SlipChip provides a practical high-throughput and rapid phenotypic method for AST and can also be used to screen different chemicals and antibiotic combinations for the treatment of multiple antibiotic-resistant bacteria.

Revealing antimicrobial resistance profile of the novel probiotic candidate Faecalibacterium prausnitzii DSM 17677

Faecalibacterium prausnitzii, a resident anaerobic bacterium commonly found in healthy gut microbiota, has been proposed as a next generation probiotic with high potential for application in food matrices and pharmaceutical formulations. Despite its recognized health benefits, detailed information regarding its antimicrobial susceptibility profile is still lacking. However, this information is crucial to determine its safety, since the absence of acquired antimicrobial resistance is required to qualify a probiotic candidate as safe for human and animal consumption. Herein, the antimicrobial susceptibility profile of F. prausnitzii DSM 17677 strain was evaluated by integrating both phenotypic and in silico data. Phenotypic antimicrobial susceptibility was evaluated by determining minimum inhibitory concentrations of 9 antimicrobials using broth microdilution and E-test® methods. Also, the whole genome of F. prausnitzii DSM 17677 was analysed, using several databases and bioinformatics tools, to identify possible antibiotic resistance genes (ARG), genomic islands (GI) and mobile genetic elements (MGE). With exception of erythromycin, the same classification (susceptible or resistant) was obtained in both broth microdilution and E-test® methods. Phenotypic resistance to ampicillin, gentamycin, kanamycin and streptomycin were detected, which was supported by the genomic context. Other ARG were also identified but they seem not to be expressed under the tested conditions. F. prausnitzii DSM 17677 genome contains 24 annotated genes putatively involved in resistance against the following classes of antimicrobials: aminoglycosides (such as gentamycin, kanamycin and streptomycin), macrolides (such as erythromycin), tetracyclines and lincosamides. The presence of putative ARG conferring resistance to β-lactams could only be detected using a broader homology search. The majority of these genes are not encoded within GI or MGE and no plasmids were reported for this strain. Despite the fact that most genes are related with general resistance mechanisms, a streptomycin-specific ARG poses the only potential concern identified. This specific ARG is encoded within a GI and a MGE, meaning that it could have been laterally acquired and might be transferred to other bacteria present in the same environment. Thus, our findings provide relevant insights regarding the phenotypic and genotypic antimicrobial resistance profiles of the probiotic candidate F. prausnitzii DSM 17677.

Phenotypic detection, antimicrobial susceptibility and virulence profile of staphylococci in the pig production setting, Makurdi, Nigeria.

Livestock, particularly pigs, have increasingly been recognized as important reservoirs for zoonotic transmission of pathogenic bacteria, including staphylococci. Livestock production systems in developing countries of sub-Saharan Africa, including Nigeria, are characterized by high misuse/abuse of antimicrobials and a close association between humans and these animals, which promotes the emergence and transmission of resistant and potentially virulent bacteria. In the present study, we investigated the occurrence and characteristics (species distribution, virulence and resistance profile) of staphylococci from smallholder backyard pig farms, slaughter slabs and pig handlers in Makurdi, Nigeria. A total of 330 nasal swabs originating from 300 pigs and 30 in-contact humans were collected and processed. One hundred and thirteen samples [34.2 %; 95 % confidence interval (CI): 29.1–39.6] comprising 103 (34.3 %; 95 % CI: 29.0–40.0) and 10 (33.3 %; 95 % CI: 17.3–52.8 %) samples from pigs and humans, respectively, were positive for staphylococci, yielding 120 isolates (pigs n=110, humans n=10). The 120 isolates were distributed into 15 species with Staphylococcus aureus (n=25) followed by Staphylococcus cohnii (n=19) and Staphylococcus sciuri (n=14) occurring more frequently. All isolates were resistant to β-lactam (100 %) antibiotics. Resistance to some critical antimicrobials, including linezolid (22 %), vancomycin (19.2 %), gentamicin (7.5%) and the fluoroquinolones ciprofloxacin (75.8 %) and enrofloxacin (66.7 %), was also observed. Majority (99.2 %) of the isolates displayed a multidrug resistance phenotype with the AMP-C-CIP-E-ENR-FOX-OX-P-S-SXT-TE phenotype being predominant. Overall, 70 % of the isolates expressed the methicillin resistance phenotype, out of which 20 % (n=17) were MRSA. Resistance to serum bactericidal activity and biofilm production were respectively observed in 45 (100 %) and 5 (11.3 %) of the coagulase-positive staphylococci. Our findings demonstrated the occurrence of a high diversity of staphylococci expressing multidrug resistance and potentially virulent phenotypes among healthy swine and pig handlers in small-scale backyard farms in North-Central Nigeria. These findings underscore the potential role of pig production settings in the emergence and dissemination of potentially virulent staphylococci and the importance of the development of antimicrobial resistance monitoring systems/implementation of control measures in developing countries. Proper hygienic practices and control of indiscriminate use and misuse of antibiotics are recommended.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains.

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

Comparison of Phenotypic and Genotypic Patterns of Antimicrobial-Resistant Bacteroides fragilis Group Isolated from Healthy Individuals in Vietnam and Japan.

PurposeNormal non-pathogenic flora can harm the host by acting as a reservoir of resistance determinants that are potentially transferable to human pathogens. This study aimed to assess the phenotypic and genotypic antimicrobial susceptibility patterns of the Bacteroides fragilis group (BFG) isolated from healthy individuals in Vietnam and Japan in order to elucidate the prevalence of antimicrobial resistance in human flora in the two economically and geographically different countries.Materials and MethodsBFG was isolated from fecal samples of 80 healthy individuals in Vietnam (n=51) and Japan (n=29). Isolated strains were identified using MALDI-TOF MS, and the minimum inhibitory concentration (MIC) of 18 antibiotics was determined using the agar dilution method. Additionally, 20 antimicrobial resistance genes were detected using standard PCR.ResultsA total of 139 BFG strains belonging to 11 BFG species were isolated from the two countries, with diversity in the prevalence of each species. B. fragilis was not the predominant species. Isolations from Vietnam and Japan showed some similarities in terms of MIC50 values, MIC90 values, and the percentage of resistant strains. However, isolations from Vietnam showed significantly higher resistance to piperacillin, cefmetazole, clindamycin, tetracycline, and minocycline. ErmB, tet36, tetM, nim, catA, and qnrA were not found in either country. CepA was more common in B. fragilis than in non-fragilis Bacteroides. In contrast, cfiA, ermG, mefA, msrSA, tetX, tetX1, bexA, qnrB, and qnrS were found only in non-fragilis Bacteroides. There were differences in the prevalence of ermG, linA, mefA, msrSA, and qnrS between isolates from Vietnam and Japan.ConclusionThis study is the first report on the antimicrobial susceptibility patterns in the BFG isolated from healthy individuals in Vietnam and Japan. Compared to isolations from Japan, isolations from Vietnam showed significantly higher resistance to antimicrobial agents. The distribution of various antibiotic resistance genes also differed between the two countries.

Antimicrobial Drug-Resistant Salmonella in Urban Cats: Is There an Actual Risk to Public Health?

The present study was undertaken to investigate the presence of Salmonella spp. in the faeces of client-owned cats in urban areas and to evaluate the risk that is posed to public health. Fresh faecal samples were collected directly from the rectums from 53 diarrhoeic and 32 non-diarrhoeic cats. The samples were individually screened for the presence of Salmonella spp. using standard methods and, in the case of positive findings, the resulting typical colonies were then biochemically confirmed using the VITEK®2 automated system. Subsequently, all of the Salmonella spp. isolates were molecularly tested for the presence of the invA gene. All of the isolates were serotyped using the slide agglutination technique according to the White–Kauffmann–Le Minor scheme. The phenotypic antimicrobial susceptibility profile of the isolated strains was obtained from the VITEK®2 system using specific cards from the Gram-negative bacteria. A total of 16 of the samples (18.82%) tested positive for Salmonella spp. according to conventional and molecular testing methods. Serotyping of the Salmonella isolates showed the presence of three serotypes, namely S. enteritidis (n = 9; 56.3%), S. typhimurium (n = 4; 25%), and S. kentucky (n = 3; 18.8%). All of the tested strains showed strong resistance towards cefazolin, cefepime, ceftazidime, and ceftriaxone. Additionally, resistance (listed in descending order of strength) was observed to trimethoprim/sulfamethoxazole (11/16; 68.8%), ampicillin (10/16; 62.5%), ampicillin/sulbactam (9/16; 56.3%), gentamicin (9/16; 56.3%), nitrofurantoin (8/16; 50.0%), and amikacin (5/16; 31.3%). No resistance was expressed against ciprofloxacin, ertapenem, imipenem, levofloxacin, piperacillin/tazobactam, and tobramycin. The results of this study highlight a substantial public health issue and medical concern, especially in vulnerable people, such as children, the elderly, and immunocompromised individuals.

Antimicrobial Use and Susceptibility of Indicator Escherichia coli in Finnish Integrated Pork Production.

In pigs, antimicrobial use (AMU) practices vary at different production phases between herds and between countries. Antimicrobial resistance (AMR) development is linked to AMU but recognized as a multi-factorial issue, and thus, any information increasing knowledge of AMU and AMR relationships is valuable. We described AMU and screened the carriage of different AMR phenotypes of indicator Escherichia coli in 25 selected Finnish piglet-producing and finishing herds that formed nine birth-to-slaughter production lines. Moreover, we studied associations between AMU and AMR in both herd types and throughout the production line. Treatment records were obtained from the national Sikava register for 1year, and AMU was quantified as mg/PCU (population correction unit) and TIs (treatment incidences). For phenotypic antimicrobial susceptibility testing, ten pen-level pooled feces samples (n=250) in each herd were collected from one room representing the oldest weaned piglets or the oldest finishing pigs. Majority of the medications (96.8%) was administered parenterally, and penicillin was the predominant antimicrobial in every herd. More different antimicrobial substances were used in piglet-producing than in finishing herds (median 5 and 1, respectively, p<0.001). As mg/PCU, sows had the highest AMU and suckling piglets had the highest TIs, whereas finishing pigs were the least treated age group. The proportion of susceptible indicator E. coli isolates of all studied isolates was 59.6%. Resistance was found most commonly against tetracycline, sulfamethoxazole, trimethoprim, and ampicillin, and multi-resistant (MR) isolates (46.5% of all resistant isolates) were resistant to a maximum of four different antimicrobials. Quinolone resistance was rare, and no resistance against 3rd-generation cephalosporins, meropenem, azithromycin, colistin, gentamicin, or tigecycline was detected. The main associations between AMU and AMR were found at antimicrobial group level when use was compared with the presence of AMR phenotypes. The proportion of resistant isolates was not associated with AMU, and herd size was not associated with either AMU or AMR. We suggest that the use of narrow-spectrum beta-lactams as a primary treatment option and lack of wide application of oral group medications potentially favors a good resistance pattern in integrated pork production.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Strains Isolated at the German Military Field Laboratory in Mazar-e Sharif, Afghanistan.

The study was performed to provide an overview of the molecular epidemiology of carbapenem-resistant Acinetobacter baumannii in Afghanistan isolated by the German military medical service during the Afghanistan conflict. A total of 18 isolates were collected between 2012 and 2018 at the microbiological laboratory of the field hospital in Camp Marmal near Mazar-e Sharif, Afghanistan, from Afghan patients. The isolates were subjected to phenotypic and genotypic differentiation and antimicrobial susceptibility testing as well as to a core genome multi-locus sequence typing (cgMLST) approach based on whole-genome next-generation sequence (wgNGS) data. Next to several sporadic isolates, four transmission clusters comprising strains from the international clonal lineages IC1, IC2, and IC9 were identified. Acquired carbapenem resistance was due to blaOXA-23 in 17/18 isolates, while genes mediating resistance against sulfonamides, macrolides, tetracyclines, and aminoglycosides were frequently identified as well. In conclusion, the assessment confirmed both the frequent occurrence of A. baumannii associated with outbreak events and a variety of different clones in Afghanistan. The fact that acquired carbapenem resistance was almost exclusively associated with blaOXA-23 may facilitate molecular resistance screening based on rapid molecular assays targeting this resistance determinant.