Antimicrobial Resistance Testing Research Articles

Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes.

ABSTRACTWith looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance.IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats “watch list” of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.

Whole-genome sequencing-based prediction and analysis of antimicrobial resistance in Yersinia enterocolitica from Ningxia, China.

Focusing on resistance trends and transmission patterns of pathogenic microorganisms is a major priority for national surveillance programs. The use of whole-genome sequencing for antimicrobial susceptibility testing (WGS-AST) is a powerful alternative to traditional microbiology laboratory methods. Yersinia enterocolitica antimicrobial resistance (AMR) in the Ningxia Hui Autonomous Region has yet to be described thoroughly in current studies. We assessed and monitored the development of Y. enterocolitica AMR in the Ningxia Hui Autonomous Region during 2007–2019 based on WGS-AST. Resistance genotypes were predicted based on WGS. Antimicrobial resistance testing using classical microbiology determined resistance to 13 antimicrobial agents in 189 Y. enterocolitica isolates from Ningxia. The highest resistance level was 97.88% for cefazolin, followed by ampicillin (AMP) (44.97%), ciprofloxacin (CIP) (25.40%), streptomycin (STR) (11.11%), and tetracycline (TET) (10.58%). Isolates emerged as chloramphenicol (CHL) and trimethoprim/sulfamethoxazole (SXT) resistant. The primary plasmid types were IncFII(Y) and ColRNAI. The TET, STR, and SXT resistance were mediated by the tetA, aph(6)-Id, aph(3″)-Ib, and sul2 genes located on the IncQ1 plasmid. The resistant strains were predominantly biotype 4/O:3/ST429 and the hosts were pigs and patients. The number of multidrug-resistant (MDR) strains was of concern, at 27.51%. At present, the prediction of antimicrobial resistance based on WGS requires a combination of phenotypes. From 2007 to 2019, Y. enterocolitica isolates from the Ningxia Hui Autonomous Region showed a relatively high rate of resistance to cefazolin (CZO) and some resistance to AMP, CIP, STR, and TET. CIP, SXT, and TET showed a relatively clear trend of increasing resistance. Plasmids carrying multiple drug resistance genes are an important mechanism for the spread of antimicrobial resistance. Isolates with low pathogenicity were more likely to present an AMR phenotype than non-pathogenic isolates.

Genetic Diversity, Antimicrobial Resistance, and Virulence Factors of Enterococcus faecalis Isolates Obtained from Stool Samples of Hospitalized Patients

Background: Enterococcus faecalis rapidly develops resistance to different antibiotics, thereby resulting in serious nosocomial infections associated with high mortality rates and different problems in the healthcare systems. Objectives: This study aimed to analyze the genetic diversity, antimicrobial resistance, and virulence factors of E. faecalis isolates obtained from the stool samples of patients in a hospital in the center of Iran. Methods: In this cross-sectional descriptive-analytical study, 108 stool samples were collected from September 2019 to February 2020 from 108 patients hospitalized in a hospital in the center of Iran. Enterococcus faecalis isolates were detected using the ddlE gene detection technique, and antimicrobial resistance testing was performed using the disc agar diffusion method. Moreover, polymerase chain reaction was used to detect antimicrobial resistance genes and virulence factors. Genetic diversity was also analyzed by enterobacterial repetitive intergenic consensus using PCR. The BioNumerics software was used to construct a dendrogram. Results: Of 108 isolates, 50 samples were E. faecalis (46.2%). The prevalence of multidrug resistance among E. faecalis isolates was 62%, and most isolates were resistant to antibiotics tetracycline (70%), erythromycin (68%), and rifampin (60%). Among the E. faecalis isolates, the most prevalent antimicrobial resistance genes were ermB (96%), aph (2′′) Ia (66%), aac(6′)-Ie (40%), and ermC (30%), and the most prevalent virulence genes were gelE (78%), asa1 (74%), and esp (74%). The genetic diversity analysis showed 25 ERIC types in two major clusters (ie, clusters H and J) and eight minor clusters (ie, clusters A-G and I). There was no significant difference between clusters H and J in terms of antimicrobial resistance and resistance genes (P > 0.05). In contrast, the prevalence of the asa1 gene was significantly higher in cluster J than in cluster H (P < 0.05). Conclusions: This study showed the high prevalence of multidrug resistance, and high heterogeneity among the E. faecalis isolates obtained from the stool samples of hospitalized patients.

Genomic analysis, antibiotic resistance, and virulence of Staphylococcus aureus from food and food outbreaks: A potential public concern

Transmission and outbreaks of Staphylococcus aureus among retail food highlights the need to comprehensive analysis the molecular characteristic of Staphylococcus aureus in foods. However, the information about Staphylococcus aureus in north China is limited. In this study, 97 and 28 S. aureus strains were isolated for analysis from 4262 samples of retail food and 61 samples food outbreaks with prevalence rate 2.28 % and 45.9 %, respectively in Jilin, China from 2014 to 2018. This study aimed to investigate the prevalence of S. aureus isolates and characterize by antimicrobial resistance testing, virulence profiles, spa typing, and multilocus sequence typing (MLST) analysis. 60 % (75/125) of the isolates contained at least enterotoxin genes including classic and new SEs genes as following: sea (40/125,32 %), see (36/125,28.8 %), sec (29/125,23.2 %), sell (29/125,23.2 %), seb (25/125,20 %), seh (22/125,17.6 %), sed (6/125,4.8 %), selq (6/125,4.8 %), and selk (6/125,4.8 %). In antimicrobial susceptibility tests, 59.2 % of the isolates (74/125) were considered as multi-drug-resistant isolates and four MRSA strains were all found with high multi-drug-resistance. Phenotype resistance to penicillin (94.4 %), erythromycin (84.2 %), clindamycin (63.9 %), and tetracycline (47.2 %) was observed which was corresponding with genotype resistance. The strains were classified to twenty-two sequence types (STs), fourteen clonal complexes (CCs), and forty-seven spa types. The predominant ST and spa types were ST1(22/125,17.6 %), ST25(20/125,16.00 %), ST398 (14/125,11.2 %) and t127 (20/125,16 %), t078 (14/125,11.2 %), t803 (7/125,5.6 %). The wgSNP analysis of these isolates in food represents showed close relatedness with food outbreaks which pose a potential health risk for consumers and warrants further attention.

Focused Commentary; About Revision of CLSI Antimicrobial Breakpoints, 2018-2021

In recent years, antimicrobial resistance has been on the rise and infectious disease specialists and other clinicians face several challenges when treating patients with antimicrobial resistant infections. Clinical and Laboratory Standards Institute (CLSI) annually publishes guidelines for antimicrobial resistance tests, including revised antimicrobial breakpoints, because of the rise in antimicrobial resistance and changes to nomenclature owing to advances in whole genome sequencing technology. However, many laboratories use historical guidelines and do not follow the revised breakpoints. Moreover, the current breakpoints have limitations. This study evaluated the changes in antimicrobial breakpoints over the last 4 years, from 2018 to 2021. Here, I describe the microbiological and clinical background of the CLSI breakpoint revision and evaluate the advantages and limitations of new breakpoints with a review of large study data. In addition, I reviewed problems associated with each antimicrobial breakpoint and made suggestions for how they might be improved, for example, increasing or decreasing the minimum inhibitory concentration (MIC) or zone diameter, deleting or adding an S, I, or R category, introducing new concepts (such as susceptible-dose dependent (SDD)), and requesting more evaluation methods. Conclusions; CLSI annually publishes guidelines for antimicrobial resistance tests. I reviewed problems associated with each antimicrobial breakpoint for last 4 years, and made suggestions for how they might be improved.

Implementation of a customised antimicrobial resistance laboratory scorecard in Cameroon, Ethiopia and Kenya.

BackgroundIn low-resource settings, antimicrobial resistance (AMR) is detected by traditional culture-based methods and ensuring the quality of such services is a challenge. The AMR Scorecard provides laboratories with a technical assessment tool for strengthening the quality of bacterial culture, identification, and antimicrobial testing procedures.ObjectiveTo evaluate the performance of the AMR Scorecard in 11 pilot laboratory evaluations in three countries also assessed with the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) checklist.MethodsPilot laboratory evaluations were conducted in Cameroon, Ethiopia and Kenya between February 2019 and March 2019. Assessors with previous SLIPTA and microbiology experience were trained. Assessors performed the laboratory assessments using the SLIPTA and AMR Scorecard tools.ResultsWeaknesses in technical procedures and the quality management systems were identified in all areas and all laboratories. Safety had the highest mean performance score (SLIPTA: 68%; AMR Scorecard: 73%) while management review had the lowest (SLIPTA: 32%; AMR Scorecard: 8%) across all laboratories. The AMR Scorecard scores were generally consistent with SLIPTA scores. The AMR Scorecard identified technical weaknesses in AMR testing, and SLIPTA identified weaknesses in the quality management systems in the laboratories.ConclusionSince the AMR Scorecard identified important gaps in AMR testing not detected by SLIPTA, it is recommended that microbiology laboratories use SLIPTA and the AMR Scorecard in parallel when preparing for accreditation. Expanding the use of the AMR Scorecard is a priority to address the need for quality clinical microbiology laboratory services in support of optimal patient care and AMR surveillance.

Impact of Pneumococcal Vaccination on Nasopharyngeal Carriage of Streptococcus pneumoniae and Microbiota Profiles in Preschool Children in South East Poland.

In 2017, Poland introduced the 10-valent pneumococcal conjugate vaccine (PCV) into its national immunization schedule. This prospective study was conducted between March and June 2020 to determine the impact of vaccination on prevalence of the nasopharyngeal carriage of S. pneumoniae in 176 healthy children and to determine how conjugate vaccines indirectly affect colonization of nasopharyngeal microbiota. Pneumococcal isolates were analyzed by serotyping and antimicrobial resistance tests. Nasopharyngeal microbiota were detected and identified using the culture method and real-time PCR amplification primers and hydrolysis-probe detection with the 16S rRNA gene as the target. In the vaccinated group of children, colonization was in 24.2% of children, compared to 21.4% in the unvaccinated group. Serotypes 23A and 23B constituted 41.5% of the isolates. Serotypes belonging to PCV10 and PCV13 constituted 4.9% and 17.1% of the isolates, respectively. S. pneumoniae isolates were resistant to penicillin (34.1%), erythromycin (31.7%), and co-trimoxazole (26.8%). Microbial DNA qPCR array correlated to increased amounts of Streptococcus mitis and S. sanguinis in vaccinated children, with reduced amounts of C. pseudodiphtericum, S. aureus, and M. catarrhalis. Introduction of PCV for routine infant immunization was associated with significant reductions in nasopharyngeal carriage of PCV serotypes and resistant strains amongst vaccine serotypes, yet carriage of non-PCV serotypes increased modestly, particularly serotype 23B.

Antimicrobial resistance of Staphylococcus aureus and Pseudomonas spp. isolated from coated skewers sold in Ouagadougou, Burkina Faso

Coated skewers are very popular in Ouagadougou. This work aim to assess the microbial quality of coated skewers sold in streets of Ouagadougou and their isolated Staphylococcus aureus and Pseudomonas spp antimicrobial resistance. A total of 30 coated skewers samples were collected from various processors for microbial analysis using standard methods. The antimicrobial resistance test was performed using the agar plate diffusion method. The microbial load varied from 6.0×104 -1.7×108 cfu/g (aerobic mesophilic bacteria (AMB)), 1.5×10-2.7×107 cfu/g (coliform), 0.0 to 6.1×101 (S. aureus) and 10-25 cfu/g (Pseudomonas spp.). Globally, 70% (21/30), 30% (09/30), 40% (12/30) and 6.67% (02/30) of the analyzed samples were unacceptable based on the load of AMB, coliform, S. aureus and Pseudomonas spp., respectively. Four coated skewers were contaminated by Pseudomonas spp. About susceptibility to antimicrobial agents, 91.67% of S. aureus strains were resistant to ceftazidime and aztreonam, while a low resistance rate was observed for the others antibiotics. Pseudomonas spp strains, were resistant to cotrimoxazol (75%) but sensitive to the other antibiotics. These results highlight poor hygienic conditions of coated skewers preparation in Ouagadougou. These practices contribute to their microbial contamination. Isolated bacteria showed different level of resistance to the tested antibiotics. There is a need of good manufacturing practices to improve the hygienic quality of coated skewers.

Methodology for laboratory-based antimicrobial resistance surveillance in animals.

Antimicrobial resistance (AMR) is a crucial and emerging multifactorial “One Health” problem involving human and animal health, agriculture, aquaculture, and environment; and posing a potential public health hazard globally. The containment of AMR justifies effective surveillance programs to explicate the magnitude of the problem across the contributing sectors. Laboratory-based AMR testing and characterization is the key component of an AMR surveillance program. An AMR surveillance program should have a “top management” for fund mobilization, planning, formulating, and multilateral coordinating of the surveillance activities. The top management should identify competent participating laboratories to form a network comprising a reference laboratory and an adequate number of sentinel laboratories. The responsibilities of the reference laboratory include the development of standardized test methods for ensuring quality and homogeneity of surveillance activities, providing training to the laboratory personnel, and in-depth AMR characterization. The sentinel laboratories will take the responsibilities of receiving samples, isolation and identification of microbes, and initial AMR characterization. The sentinel laboratories will use simple antimicrobial susceptibility test (AST) methods such as disk diffusion tests, whereas the reference laboratories should use automated quantitative AST methods as well as advanced molecular methods to explicit AMR emergence mechanisms. Standard guidelines set by Clinical Laboratory Standards Institute or the European Committee on Antimicrobial Susceptibility Testing, should be followed to bring about conformity and harmonization in the AST procedures. AMR surveillance program in animals is eventually similar to that in human health with the exception is that veterinary antibiotics and veterinary pathogens should be given preference here. Hence, the review study was envisaged to look deep into the structure of the AMR surveillance program with significance on laboratory-based AMR testing and characterization methods.

Bovine mastitis in northeastern Brazil: Occurrence of emergent bacteria and their phenotypic and genotypic profile of antimicrobial resistance

The aim of this study was to identify emergent pathogens associated with bovine mastitis in northeastern Brazil and to characterize them for phenotypic and genotypic resistance to antimicrobials. A total of 321 milk samples from cows with subclinical mastitis were collected, and the isolates obtained in culture were identified using matrix-associated laser desorption-ionization - time of flight mass spectrometry. Phenotypic and genotypic antimicrobial resistance tests were performed. We identified 72 bacteria considered emergent in the study region: Enterococcus faecalis (26.3%; 19/72), Streptococcus agalactiae (22.2%; 16/72), Enterococcus faecium (20.0%; 15/72), Escherichia coli (6.9%; 5/72), 6.9% (5/72) Lactococcus garvieae (6.9%; 5/72), Acinetobacter baumannii (5.5%; 4/72), Bacillus subtilis (1.3%; 1/72), Kocuria marina (1.3%; 1/72), Macrococcus caseolyticus (1.3%; 1/72), Microbacterium resistens (1.3%; 1/72), Micrococcus luteus (1.3%; 1/72), Streptococcus dysgalactiae (1.3%; 1/72), Streptococcus hyovaginalis (1.3%; 1/72) and Streptococcus pluranimalium (1.3%; 1/72). The antibiogram revealed the following resistance profiles: ampicillin (77.7%; 56/72), cefoxitin (69.4%; 50/72), erythromycin (61.1%; 44/72), oxacillin (63.8%; 46/72), penicillin (79.1%; 57/72), tetracycline (63.8%; 46/72), gentamicin (25.0%; 18/72), and vancomycin (20.8%; 15/72). Of the isolates, 83.4% (60/72) showed multiple resistance to antimicrobials. The tetM gene was identified in 43.0% (31/72) of the isolates, followed by tetL (31.9%; 23/72), and blaZ (26.3%; 19/72). 83.4% (60/72) of the isolates presented a multiple antimicrobial resistance index higher than 0,2. Emergent bacteria with zoonotic and multiresistant potential occur in cows with mastitis in northeastern Brazil. It is necessary to monitor the occurrence of these and other bacteria in livestock environments and develop control strategies to prevent their spread.

Magnitude of Drug-Resistant Gram-Positive Bacterial Pathogens, and Its Associated Factors from External Ocular Infected Patients Attending at Jinka General Hospital Ophthalmic Clinic, Southern Ethiopia.

BackgroundThe emergence and the incidence of antimicrobial-resistant Gram-positive bacteria increase the risk of treatment failure of ocular infection. Thus, this study aimed to determine the magnitude of drug-resistant Gram-positive bacterial pathogens and associated factors among patients attending Jinka General Hospital Ophthalmic Clinic in Southern Ethiopia.MethodsA facility-based cross-sectional study was conducted on 347 external ocular infected patients attending Jinka General Hospital Ophthalmic Clinic from 15th March to 20th June 2021. Study participants were selected by a systematic random sampling technique. Required data were collected by using structured questionnaires. Swabs of the external eye were obtained with a sterile cotton swab and processed in the Jinka branch Public Health Laboratory. Each sample was inoculated on blood agar, chocolate agar, and mannitol salt agar and incubated aerobically and micro-aerobically at 37°C for 48 hrs. Identification was done by standard microbiological protocols and antimicrobial resistance testing by Kirby Bauer’s disk diffusion technique. Logistic regression was used to identify the associated factors with Gram-positive bacterial external-ocular infection.ResultsThe overall prevalence of Gram-positive bacterial pathogens among external ocular samples was 119/347 (34.3%). S. aureus 57/119 (47.39%), followed by coagulase-negative Staphylococcus spp. 38/119 (31.9%), and S. pneumoniae 13/119 (10.9%) were predominantly isolated. Overall, multi-drug resistance was observed in 72/119 (60.5%) of the bacteria isolates. The 78.9% of isolated S. aureus were MDR. Methicillin-resistant S. aureus (MRSA) and Methicillin-resistant CoNS (MR-CoNS) were accounted for 45.6% and 36.8%, respectively. Previous use of ocular antibiotics was statistically associated to external-ocular Gram-positive bacterial infection [AOR= 1.624, 95% CI: (1.037–2.542)].ConclusionHigh levels of drug resistance were observed for commonly prescribed antibiotics, which attracted the attention of an ophthalmic clinic. Thus, for the effective treatment and management of bacterial eye infections, regular monitoring of drug resistance trends is essential.

Prevalence, Serotype Distribution and Antimicrobial Resistance of Non-Typhoidal Salmonella in Hospitalized Patients in Conghua District of Guangzhou, China.

Salmonella infection is a major public health concern worldwide, has contributed to an increased economic burden on the health systems. Non-typhoidal Salmonella (NTS) is a common cause of bacterial enteritis in humans, causing 93.8 million cases of gastroenteritis globally each year, with 155,000 deaths. Guangzhou city is situated in the south of China and has a sub-tropical climate, the heat and heavy rainfall helps the spread of NTS. However, no information of NTS infection is available in humans in Conghua District, the largest administrative district of Guangzhou. To understand the prevalence, serotype distribution, risk factors and drug resistance of NTS infection in humans in the survey area, an epidemiological investigation was conducted in hospitalized patients in Conghua District in Guangzhou, China. A total of 255 fecal specimens were collected from hospitalized patients (one each), with a questionnaire for each participant, and NTS infection was identified by culture, as well as serotypes confirmed by slide agglutination tests. An average prevalence of 20.39% (52/255) was observed and three serogroups were identified—serogroup B (n = 46), serogroup C1 (n = 4) and serogroup D1 (n = 2). Among them, Salmonella Typhimurium (n = 39) was the most common serotype. Children aged <3 years were observed to have a statistically higher prevalence of NTS infection than adults (25.15% versus 4.65%, P = 0.006); children with artificial feeding had a statistically higher prevalence than those with breastfeeding (30.77% versus 8.33%, P = 0.044). Antimicrobial resistance testing revealed that the majority of strains were resistant to ampicillin (92.16%), as well as 47.06% of all strains were multi-drug resistant. Therefore, it is necessary to continuous monitoring and rational use of antibiotics, which will be helpful to reduce the prevalence of resistant strains. These data will aid in making efficient control strategies to intervene with and prevent occurrence of salmonellosis.

Antimicrobial Resistance Surveillance of Pigs and Chickens in the Lao People's Democratic Republic, 2018-2021.

The use of antimicrobials in the livestock sector has been identified as a driver for the emergence of antimicrobial resistance (AMR), and AMR has become a growing public health and economic threat in the Lao PDR. We conducted surveillance for AMR in five provinces of the Lao PDR, in order to determine the antimicrobial susceptibility of Escherichia coli and Salmonella spp. isolated from caecal samples from slaughtered pigs at slaughterhouses and from slaughtered chickens at markets during two different time periods: 2018/2019 and 2020/2021. Antimicrobial susceptibility was determined using a panel of 14 antimicrobials using the broth microdilution technique. E. coli and Salmonella from chickens (62% and 33%, respectively) and pigs (88% and 81%, respectively) exhibited resistance to ≥3 classes of antimicrobials. Of important public health concern was the detection of Salmonella resistant to cefotaxime/ceftazidime, ciprofloxacin, and colistin, deemed as critically important antimicrobials in human medicine. This study aimed to evaluate a national sampling strategy at slaughterhouses and wet markets, and to pilot the laboratory methodologies for bacterial recovery and AMR testing. Experiences from this study will inform capacity development for a national AMR surveillance program, and these early data could serve as reference points for monitoring the impact of the Lao PDR’s national action plan to contain AMR.

Short-term and long-term effects of antimicrobial use on antimicrobial resistance in broiler and turkey farms

ABSTRACT Antimicrobials have been widely used in poultry, promoting antimicrobial resistance (AMR) emergence and spread. Resistant bacteria selected by antimicrobial use (AMU) can contaminate the farm environment and transfer resistance genes to other bacteria, providing opportunities for persistence and (re-)colonization of subsequent flocks and potentially jeopardizing antimicrobial treatments. We investigated the effects of AMU on AMR in poultry in the long-term (due to historical AMU in the farm) and in the short-term (due to current AMU in a flock). Litter samples from 35 broiler and 35 turkey farms in North-East Italy were sampled longitudinally for AMR testing of E. coli indicator bacteria in 2019/2020. Differences in AMR as a function of historical AMU (Defined Daily Doses in 2016–2018), current AMU in the sampled flock, farm size and season were tested using Generalized Estimating Equation regression analysis. In both broilers and turkeys, the highest resistance levels were observed for sulfamethoxazole (>70%), followed by ampicillin (54–60%). Only a few positive associations between historical levels of penicillin use and the specific resistance levels to penicillin in broiler farms, and the overall historical AMU and resistance to trimethoprim in turkey flocks, were significant. Current AMU showed significant effects on resistance to sulfamethoxazole, trimethoprim, ciprofloxacin and tetracycline in turkey flocks. Significant effects of farm size on some AMR levels were also identified. We found a stronger association between current AMU and AMR compared to historical AMU and AMR. AMR persistence in the farm environment in the absence of direct AMU pressure needs to be further investigated. RESEARCH HIGHLIGHTS Sulfamethoxazole and ampicillin resistance are the highest in E. coli isolates of both broilers and turkeys. Short-term effects of antibiotic use on resistance are seen more often than long-term effects. Historical penicillin use is associated with penicillin resistance in broilers. There is an association between historical use and resistance to trimethoprim in turkeys.

First National Genomic Epidemiological Study of Neisseria gonorrhoeae Strains Spreading Across Sweden in 2016.

The increasing transmission and antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health concern with worrying trends of decreasing susceptibility to also the last-line extended-spectrum cephalosporin (ESC) ceftriaxone. A dramatic increase of reported gonorrhea cases has been observed in Sweden from 2016 and onward. The aim of the present study was to comprehensively investigate the genomic epidemiology of all cultured N. gonorrhoeae isolates in Sweden during 2016, in conjunction with phenotypic AMR and clinical and epidemiological data of patients. In total, 1279 isolates were examined. Etest and whole-genome sequencing (WGS) were performed, and epidemiological data obtained from the Public Health Agency of Sweden. Overall, 51.1%, 1.7%, and 1.3% resistance to ciprofloxacin, cefixime, and azithromycin, respectively, was found. No isolates were resistant to ceftriaxone, however, 9.3% of isolates showed a decreased susceptibility to ceftriaxone and 10.5% to cefixime. In total, 44 penA alleles were found of which six were mosaic (n = 92). Using the typing schemes of MLST, NG-MAST, and NG-STAR; 133, 422, and 280 sequence types, respectively, and 93 NG-STAR clonal complexes were found. The phylogenomic analysis revealed two main lineages (A and B) with lineage A divided into two main sublineages (A1 and A2). Resistance and decreased susceptibility to ESCs and azithromycin and associated AMR determinants, such as mosaic penA and mosaic mtrD, were predominantly found in sublineage A2. Resistance to cefixime and azithromycin was more prevalent among heterosexuals and MSM, respectively, and both were predominantly spread through domestic transmission. Continuous surveillance of the spread and evolution of N. gonorrhoeae, including phenotypic AMR testing and WGS, is essential for enhanced knowledge regarding the dynamic evolution of N. gonorrhoeae and gonorrhea epidemiology.

Advances in antimicrobial resistance testing.

Antimicrobial resistance (AMR), especially bacterial AMR, poses a global threat to public health and has become a huge obstacle to the effective control of related infectious diseases. Following the golden age of antimicrobials discovery between the 1940s and 1960s, antimicrobial abuse resulted in the rapid emergence of AMR. Nowadays, the problem of AMR has become increasingly serious, and some bacteria have reached the brink of no suitable antimicrobials available. Rapid detection of AMR and level quantification are the prerequisites to control the spread of AMR. Although time-consuming, traditional phenotype-based methods are still the primary methods used in clinical laboratories and are regarded as the gold standard for AMR identification. To offset the limitation of the long turnaround time of phenotype-based methods, molecular detection methods such as polymerase chain reaction (PCR), isothermal amplification, high-throughput sequencing, gene microarray, and mass spectrometry have begun to be widely used and served as important complements to phenotype-based methods. This chapter will describe the advances in the above technologies applied in AMR testing.

LAP-MALDI MS coupled with machine learning: an ambient mass spectrometry approach for high-throughput diagnostics.

Large-scale population screening for early and accurate detection of disease is a key objective for future diagnostics. Ideally, diagnostic tests that achieve this goal are also cost-effective, fast and easily adaptable to new diseases with the potential of multiplexing. Mass spectrometry (MS), particularly MALDI MS profiling, has been explored for many years in disease diagnostics, most successfully in clinical microbiology but less in early detection of diseases. Here, we present liquid atmospheric pressure (LAP)-MALDI MS profiling as a rapid, large-scale and cost-effective platform for disease analysis. Using this new platform, two different types of tests exemplify its potential in early disease diagnosis and response to therapy. First, it is shown that LAP-MALDI MS profiling detects bovine mastitis two days before its clinical manifestation with a sensitivity of up to 70% and a specificity of up to 100%. This highly accurate, pre-symptomatic detection is demonstrated by using a large set of milk samples collected weekly over six months from approximately 500 dairy cows. Second, the potential of LAP-MALDI MS in antimicrobial resistance (AMR) detection is shown by employing the same mass spectrometric setup and similarly simple sample preparation as for the early detection of mastitis.

Antimicrobial resistance in Libya: A systematic literature review of two decades

Background: In low- and middle-income countries, antimicrobial resistance (AMR) imposes a significant burden on patients and health-care systems. Due to a lack of data, the overall impact of AMR in Libya is not well known or documented. As a result, this study presents the results of a review of available data on AMR in Libya over the past 20 years (from 2002 to 2021) to aid understanding of the current AMR situation in this portion of the continent. Methods: Articles related to the topic were researched using databases and search engines such as PubMed, Google Scholar, and ResearchGate websites. These articles were selected based on predetermined inclusion and exclusion criteria. The total number of tested isolates for each of the reported Bacterial spp. was used to calculate antibiotic resistance to a specific bacterium. Results: Studies published in the past 20 years, representing reports of 18,160 AMR tests, showed that Urinary Tract Infection (UTI) was the most reported clinical diagnosis in Libya (61.3%). Out of 43 articles, Staphylococcus aureus was the most common Gram-positive bacteria documented in (31, 61.3%) studies, and was most common in 59.78% of skin infections. Whereas, Pseudomonas spps., were the most common Gram-negative bacteria presented in (23, 53.48%) studies, and were commonly isolated in respiratory infection (9.39%). Among Gram-negative bacteria, Pseudomonas spp. reported a high resistance percentage for penicillin beta-lactam antibiotic, i.e., piperacillin (10.4%) and to the first-generation cephalosporins antibiotics, i.e., cefazolin (7.7%). However, they are susceptible to metronidazole, vancomycin, and colistin. Gram-positive bacteria, S. aureus shows high resistance to oxacillin, followed by gentamycin and cefoxitin (8.5%, 8.3%, and 8.3%, respectively). Effective antibiotics against S. aureus were azithromycin, clarithromycin, and metronidazole, whose susceptibility was 99.9% each, while 1% of S. aureus were vancomycin-resistant S. aureus. Conclusion: This study gives a comprehensive analysis of the state of AMR in Libya with respect to the most regularly prescribed antibiotics. The findings of the research show the alarmingly persistent occurrences of AMR in Libya, as well as the critical need to establish national action, plans to combat AMR and improve surveillance programs.

Antibiotic Susceptibility Pattern and Identification of Multidrug Resistant Novel &amp;lt;i&amp;gt;Salmonella&amp;lt;/i&amp;gt; Strain in Poultry Chickens of Hathazari Region in Chattogram, Bangladesh

Poultry chickens are potential source of transmission of zoonotic Salmonella, into human food chain, causing food-borne illness and also hindering development of poultry industry in Bangladesh. The non-judicious uses of antibiotics in poultry farm have increased the multidrug resistant bacteria. So, this study reports the occurrence of Salmonella in poultry samples (meat, egg, liver and cloacal swab) and the antimicrobial resistance pattern of the isolates. This study was carried out throughout the period of May 2019-March 2020, at the bacteriological laboratory in the Department of Microbiology, University of Chittagong. Isolates were identified on the basis of cultural and biochemical tests from a total of 25 broiler samples (meat, liver, eggshell and cloacal swab). Antibiotic susceptibility pattern was observed using Kirby-Bauer disk diffusion method. The overall detection rate of Salmonella was 48% (12/25) and the highest occurrence was noticed in raw meat 62.5% and the lowest in liver (37.5%). The antimicrobial resistance tests revealed that all the isolates (n = 12) exhibited 100% resistance to vancomycin and cephalexin, followed by ampicillin (75%), nalidixic acid (58.33%), chloramphenicol (41.66%), doxycycline (50%), and neomycin (50%). On the other hand, ciprofloxacin showed 83.33%, ceftazidime and amoxicillin showed 91.6% sensitivity respectively. A considerably high proportion of isolates (11/12, 91.67%) was resistant to three or more antibiotics and 6 multidrug profiles were observed. The ampicillin-chloramphenicol-nalidixic acid-neomycin-cephalexin-doxycycline-vancomycin (4/12) was more frequently observed phenotype in multidrug profiles. Finally, two multidrug-resistant strains of Salmonella were identified and classified based on their 16S ribosomal RNA gene sequences as Salmonella enterica subsp. enterica strain Eshaa2 and Salmonella enterica subsp. enterica strain Eshiika3 at NCBI GenBank with Accession no. MT163513 and MT164531 respectively. So, more attention should be focused on increasing antibiotic surveillance to cope with the spread of emerging resistance and on the alternative approaches.

Direct antimicrobial resistance prediction from clinical MALDI-TOF mass spectra using machine learning.

Early use of effective antimicrobial treatments is critical for the outcome of infections and the prevention of treatment resistance. Antimicrobial resistance testing enables the selection of optimal antibiotic treatments, but current culture-based techniques can take up to 72 hours to generate results. We have developed a novel machine learning approach to predict antimicrobial resistance directly from matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra profiles of clinical isolates. We trained calibrated classifiers on a newly created publicly available database of mass spectra profiles from the clinically most relevant isolates with linked antimicrobial susceptibility phenotypes. This dataset combines more than 300,000 mass spectra with more than 750,000 antimicrobial resistance phenotypes from four medical institutions. Validation on a panel of clinically important pathogens, including Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, resulting in areas under the receiver operating characteristic curve of 0.80, 0.74 and 0.74, respectively, demonstrated the potential of using machine learning to substantially accelerate antimicrobial resistance determination and change of clinical management. Furthermore, a retrospective clinical case study of 63 patients found that implementing this approach would have changed the clinical treatment in nine cases, which would have been beneficial in eight cases (89%). MALDI-TOF mass spectra-based machine learning may thus be an important new tool for treatment optimization and antibiotic stewardship.