Optimizing a Screening Protocol for Potential Extended-Spectrum ß-Lactamase Escherichia coli on MacConkey Agar for Use in a Global Surveillance Program.

The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is worrisome. Coordinated efforts to better understand global prevalence and risk factors are needed. Developing lower- and middle-income countries need reliable, readily available, and cost-effective solutions for detecting ESBL E. coli to contribute to global surveillance. We evaluated MacConkey agar supplemented with ceftriaxone or cefotaxime as a screening method for accurately detecting and quantifying potential ESBL E. coli MacConkey agar from eight manufacturers, representing seven countries, was prepared with 2 or 4 µg/ml ceftriaxone or cefotaxime. Four E. coli strains (NC11, ATCC 25922, CM-13457, and CM-10455) and one Klebsiella pneumoniae strain (CM-11073) were grown overnight, serially diluted, and plated in triplicate for enumeration on all medium combinations. After recovery was assessed, US-1 MacConkey agar with cefotaxime was used to further evaluate the reproducibility and detection of potential ESBL E. coli from poultry cecal (n = 30) and water (n = 30) samples. Results indicated the recovery of E. coli 13457 from four MacConkey agar manufacturers was reduced by up to 4 log CFU/ml, and phenotypic differences in colony size and color were apparent for each manufacturer for control E. coli strains. A true ESBL, NC11, was not reduced with 4 µg/ml cefotaxime. From ceca and water, potential ESBL E. coli isolates were only confirmed from MacConkey agar with 4 µg/ml cefotaxime, where 45% and 16.6% of E. coli isolates phenotypically expressed ESBL production. The quality and reproducibility of MacConkey agar varied by manufacturer, which suggests that a single manufacturer and medium type should be selected for global monitoring efforts so that training and interpretation can be standardized.

Extended spectrum beta-lactamase and fluoroquinolone resistance genes among Escherichia coli and Salmonella isolates from children with diarrhea, Burkina Faso.

BACKGROUND: The emergence and spread of multidrug-resistant gram-negative bacteria (MDR) has become a major public health concern worldwide. This resistance is caused by enzymes-mediated genes (i.e., extended spectrum beta-lactamases) that are common in certain Enterobacterioceae species. However, the distribution of these genes is poorly documented in Burkina Faso. This study aims to determine the prevalence and distribution of the resistant genes coding for broad spectrum beta-lactamases and quinolones in rural Burkina Faso. METHODS: Multiplex PCR assays were carried out to detect ESBL-encoding genes, including blaOXA, blaTEM, blaCTX-M, blaSHV. The assays also assessed the presence of quinolone resistance gene namely qnrA, qnrB and qnrS in the quinolone-resistance DEC and Salmonella strains. RESULTS: The Extended-Spectrum Beta-Lactamases (ESBL) resistance phenotype was reported in all the E. coli isolates (5/5). Cross-resistance phenotype to quinolones (CRQ) was shown by one Salmonella strain (1/9) and three E. coli (3/5). Cross-resistance phenotypes to fluoroquinolones (CRFQ) were harboured by one Salmonella (1/9) and carbapenemase phenotypes were detected in two E. coli strains (2/5). Whilst the blaOXA genes were detected in 100% (5/5) of E. coli isolates and in 33.33% (3/9) Salmonella isolates. One strain of E. coli (1/5) harbored the blaCTX-M gene and the qnrB gene simultaneously. CONCLUSIONS: This study identified ß-lactam (bla) and quinolone resistance (qnr) genes in multidrug-resistant E. coli and Salmonella spp. in rural Burkina Faso. Our finding which highlighted the enterobacteriaceae strains resistance to ß-lactams and quinolones are of high interest for adequate management of antimicrobial resistant genes outbreak in Burkina Faso.

Phenotypic and Genotypic Characterization of Escherichia coli and Salmonella enterica from Dairy Cattle Farms in the Wakiso District, Uganda: A Cross-Sectional Study.

Enterobacteriaceae producing ß-lactamases have spread rapidly worldwide and pose a serious threat to human-animal-environment interface. In this study, we present the presence of Salmonella enterica (1.3%) and commensal Escherichia coli (96.3%) isolated from 400 environmental fecal dairy cattle samples over 20 farms in Uganda. Among E. coli isolates, 21% were resistant to at least one antimicrobial tested and 7% exhibited multidrug resistance. Four E. coli isolates displayed extended-spectrum beta-lactamase (ESBL)-producing genes, including blaCTX-M-15 (n = 2/4), blaCTX-M-27 (n = 1/4), blaSHV-12 (n = 1/4), and blaTEM-1B (n = 2/4). Whole genome sequencing confirmed the presence of the plasmid-mediated quinolone resistance qnrS1 gene among three ESBL isolates. No statistically significant differences in seasonal prevalence for E. coli and S. enterica among dairy cattle sampling periods were observed. Furthermore, to our knowledge, this is the first report of E. coli carrying blaCTX-M-15, blaCTX-M-27, blaSHV-12, or qnrS1 isolated from dairy cattle in Uganda. We conclude that the presence of globally disseminated blaCTX-M-15 and blaCTX-M-27 warrants further study to prevent further spread. In addition, the presence of fluoroquinolone resistant ESBL-producing E. coli on dairy farms highlights the potential risk among the human-livestock-environment interaction. This study can be used as a baseline for implementation of a more robust national integrated surveillance system throughout Uganda.

Proficiency of WHO Global Foodborne Infections Network External Quality Assurance System Participants in Identification and Susceptibility Testing of Thermotolerant Campylobacter spp. from 2003 to 2012.

Campylobacter spp. are foodborne and waterborne pathogens. While rather accurate estimates for these pathogens are available in industrialized countries, a lack of diagnostic capacity in developing countries limits accurate assessments of prevalence in many regions. Proficiency in the identification and susceptibility testing of these organisms is critical for surveillance and control efforts. The aim of the study was to assess performance for identification and susceptibility testing of thermotolerant Campylobacter spp. among laboratories participating in the World Health Organization (WHO) Global Foodborne Infections Network (GFN) External Quality Assurance System (EQAS) over a 9-year period. Participants (primarily national-level laboratories) were encouraged to self-evaluate their performance as part of continuous quality improvement. The ability to correctly identify Campylobacter spp. varied by year and ranged from 61.9% (2008) to 90.7% (2012), and the ability to correctly perform antimicrobial susceptibility testing (AST) for Campylobacter spp. appeared to steadily increase from 91.4% to 93.6% in the test period (2009 to 2012). The poorest performance (60.0% correct identification and 86.8% correct AST results) was observed in African laboratories. Overall, approximately 10% of laboratories reported either an incorrect identification or antibiogram. As most participants were supranational reference laboratories, these data raise significant concerns regarding capacity and proficiency at the local clinical level. Addressing these diagnostic challenges is critical for both patient-level management and broader surveillance and control efforts.

World Health Organization Ranking of Antimicrobials According to Their Importance in Human Medicine: A Critical Step for Developing Risk Management Strategies to Control Antimicrobial Resistance From Food Animal Production.

Antimicrobial use in food animals selects for antimicrobial resistance in bacteria, which can spread to people. Reducing use of antimicrobials-particularly those deemed to be critically important for human medicine-in food production animals continues to be an important step for preserving the benefits of these antimicrobials for people. The World Health Organization ranking of antimicrobials according to their relative importance in human medicine was recently updated. Antimicrobials considered the highest priority among the critically important antimicrobials were quinolones, third- and fourth-generation cephalosporins, macrolides and ketolides, and glycopeptides. The updated ranking allows stakeholders in the agriculture sector and regulatory agencies to focus risk management efforts on drugs used in food animals that are the most important to human medicine. In particular, the current large-scale use of fluoroquinolones, macrolides, and third-generation cephalosporins and any potential use of glycopeptides and carbapenems need to be addressed urgently.

WHO's essential medicines and AWaRe: recommendations on first- and second-choice antibiotics for empiric treatment of clinical infections.

The WHO Model List of Essential Medicines (EML) prioritizes medicines that have significant global public health value. The EML can also deliver important messages on appropriate medicine use. Since 2017, in response to the growing challenge of antimicrobial resistance, antibiotics on the EML have been reviewed and categorized into three groups: Access, Watch, and Reserve, leading to a new categorization called AWaRe. These categories were developed taking into account the impact of different antibiotics and classes on antimicrobial resistance and the implications for their appropriate use. The 2023 AWaRe classification provides empirical guidance on 41 essential antibiotics for over 30 clinical infections targeting both the primary health care and hospital facility setting. A further 257 antibiotics not included on the EML have been allocated an AWaRe group for stewardship and monitoring purposes. This article describes the development of AWaRe, focussing on the clinical evidence base that guided the selection of Access, Watch, or Reserve antibiotics as first and second choices for each infection. The overarching objective was to offer a tool for optimizing the quality of global antibiotic prescribing and reduce inappropriate use by encouraging the use of Access antibiotics (or no antibiotics) where appropriate. This clinical evidence evaluation and subsequent EML recommendations are the basis for the AWaRe antibiotic book and related smartphone applications. By providing guidance on antibiotic prioritization, AWaRe aims to facilitate the revision of national lists of essential medicines, update national prescribing guidelines, and supervise antibiotic use. Adherence to AWaRe would extend the effectiveness of current antibiotics while helping countries expand access to these life-saving medicines for the benefit of current and future patients, health professionals, and the environment.

Identification of CTX-M Type ESBL E. coli from Sheep and Their Abattoir Environment Using Whole-Genome Sequencing.

Widespread dissemination of extended-spectrum beta-lactamase (ESBL) Escherichia coli (E. coli) in animals, retail meats, and patients has been reported worldwide except for limited information on small ruminants. Our study focused on the genotypic characterization of ESBL E. coli from healthy sheep and their abattoir environment in North Carolina, USA. A total of 113 ESBL E. coli isolates from sheep (n = 65) and their abattoir environment (n = 48) were subjected to whole-genome sequencing (WGS). Bioinformatics tools were used to analyze the WGS data. Multiple CTX-M-type beta-lactamase genes were detected, namely blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-32, blaCTX-M-55, and blaCTX-M-65. Other beta-lactamase genes detected included blaCMY-2, blaTEM-1A/B/C, and blaCARB-2. In addition, antimicrobial resistance (AMR) genes and/or point mutations that confer resistance to quinolones, aminoglycosides, phenicols, tetracyclines, macrolides, lincosamides, and folate-pathway antagonists were identified. The majority of the detected plasmids were shared between isolates from sheep and the abattoir environment. Sequence types were more clustered around seasonal sampling but dispersed across sample types. In conclusion, our study reported wide dissemination of ESBL E. coli in sheep and the abattoir environment and associated AMR genes, point mutations, and plasmids. This is the first comprehensive AMR and WGS report on ESBL E. coli from sheep and abattoir environments in the United States.

International lineages of Salmonella enterica serovars isolated from chicken farms, Wakiso District, Uganda.

The growing occurrence of multidrug-resistant (MDR) Salmonella enterica in poultry has been reported with public health concern worldwide. We reported, recently, the occurrence of Escherichia coli and Salmonella enterica serovars carrying clinically relevant resistance genes in dairy cattle farms in the Wakiso District, Uganda, highlighting an urgent need to monitor food-producing animal environments. Here, we present the prevalence, antimicrobial resistance, and sequence type of 51 Salmonella isolates recovered from 379 environmental samples from chicken farms in Uganda. Among the Salmonella isolates, 32/51 (62.7%) were resistant to at least one antimicrobial, and 10/51 (19.6%) displayed multiple drug resistance. Through PCR, five replicon plasmids were identified among chicken Salmonella isolates including IncFIIS 17/51 (33.3%), IncI1α 12/51 (23.5%), IncP 8/51 (15.7%), IncX1 8/51 (15.7%), and IncX2 1/51 (2.0%). In addition, we identified two additional replicons through WGS (Whole Genome Sequencing; ColpVC and IncFIB). A significant seasonal difference between chicken sampling periods was observed (p = 0.0017). We conclude that MDR Salmonella highlights the risks posed to animals and humans. Implementing a robust, integrated surveillance system will aid in monitoring MDR zoonotic threats.

Role of deoxyribose catabolism in colonization of the murine intestine by pathogenic Escherichia coli strains.

We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

World Health Organization ranking of antimicrobials according to their importance in human medicine: A critical step for developing risk management strategies for the use of antimicrobials in food production animals.

The use of antimicrobials in food animals creates an important source of antimicrobial-resistant bacteria that can spread to humans through the food supply. Improved management of the use of antimicrobials in food animals, particularly reducing the usage of those that are "critically important" for human medicine, is an important step toward preserving the benefits of antimicrobials for people. The World Health Organization has developed and applied criteria to rank antimicrobials according to their relative importance in human medicine. Clinicians, regulatory agencies, policy makers, and other stakeholders can use this ranking when developing risk management strategies for the use of antimicrobials in food production animals. The ranking allows stakeholders to focus risk management efforts on drugs used in food animals that are the most important to human medicine and, thus, need to be addressed most urgently, such as fluoroquinolones, macrolides, and third- and fourth-generation cephalosporins.

Results of use of WHO Global Salm-Surv external quality assurance system for antimicrobial susceptibility testing of Salmonella isolates from 2000 to 2007.

An international External Quality Assurance System (EQAS) for antimicrobial susceptibility testing of Salmonella was initiated in 2000 by the World Health Organization (WHO) Global Salm-Surv in order to enhance the capacities of national reference laboratories to obtain reliable data for surveillance purposes worldwide. Seven EQAS iterations have been conducted from 2000 to 2007. In each iteration, participating laboratories submitted susceptibility results from 10 to 15 antimicrobial agents for eight Salmonella isolates and an Escherichia coli reference strain (ATCC 25922). A total of 287 laboratories in 102 countries participated in at least one EQAS iteration. A large number of laboratories reported results for the E. coli ATCC 25922 reference strain which were outside the quality control ranges. Critical deviations for susceptibility testing of the Salmonella isolates varied from 4% in 2000 to 3% in 2007. Consistent difficulties were observed in susceptibility testing of amoxicillin-clavulanic acid, cefotaxime, ceftazidime, streptomycin, sulfonamides, and tetracycline. Regional variations in performance were observed, with laboratories in central Asia, Africa, and the Middle East not performing as well as those in other regions. Results from the WHO Global Salm-Surv EQAS show that most laboratories worldwide are capable of correctly performing antimicrobial susceptibility testing of Salmonella isolates, but they also indicate that further improvement for some laboratories is needed. In particular, further training and dissemination of information on quality control, appropriate interpretive criteria (breakpoints), and harmonization of the methodology worldwide through WHO Global Salm-Surv and other programs will contribute to the generation of comparable and reliable antimicrobial susceptibility data.

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Antimicrobial resistance: A round table discussion on the "One Health" concept from the Gulf Cooperation Council Countries. Part One: A focus on Leadership.

The threat of Antimicrobial Resistance (AMR) has attracted the highest level of attention after the United Nation (UN) General Assembly's High Level meeting on AMR in 2016. During that meeting a temporary committee, the inter agency coordination group (IACG) [1] was formulated, under the chairmanship of the Director General of the World Health Organization (DG-WHO) and the UN Deputy Secretary General. The IACG group was tasked to link, guide and advise on political steps needed to address and mitigate the emergence of AMR globally [2]. Efforts to combat AMR, however, have previously been developed prior to this initiative by several decades. Yet, the emergence of resistance continues to be on the rise. The Gulf Cooperation Council Center for Infection Control (GCC-IC) was been established in 2005 and has made major efforts to address all aspects related to human infection control in the member countries. Many of which have been reached. In 2015, the board was able to draft through its multidisciplinary meeting the first roadmap for the counties on this issue [3]. Two years later a follow-up meeting took place in Riyadh on April 2017. Regional and international experts joined in to share and contribute to the understanding of AMR and to advise on further steps needed for this initiative. We dedicated 3 papers on this meeting. The first paper is to provide an update on progress form the GCC countries and further steps that need to be taken, (in press). The second paper is part one out of two round table discussions. The first round table discussion paper, which is the current paper, addresses AMR and the "One Health" concept with a focus on leadership. The second round table discussion paper addresses AMR and the "One Health" concept with a focus on human health [4].

World Health Organization (WHO) guidelines on use of medically important antimicrobials in food-producing animals.

Background: Antimicrobial use in food-producing animals selects for antimicrobial resistance that can be transmitted to humans via food or other transmission routes. The World Health Organization (WHO) in 2005 ranked the medical importance of antimicrobials used in humans. In late 2017, to preserve the effectiveness of medically important antimicrobials for humans, WHO released guidelines on use of antimicrobials in food-producing animals that incorporated the latest WHO rankings. Methods: WHO commissioned systematic reviews and literature reviews, and convened a Guideline Development Group (GDG) of external experts free of unacceptable conflicts-of-interest. The GDG assessed the evidence using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, and formulated recommendations using a structured evidence-to-decision approach that considered the balance of benefits and harms, feasibility, resource implications, and impact on equity. The resulting guidelines were peer-reviewed by an independent External Review Group and approved by the WHO Guidelines Review Committee. Results: These guidelines recommend reductions in the overall use of medically important antimicrobials in food-producing animals, including complete restriction of use of antimicrobials for growth promotion and for disease prevention (i.e., in healthy animals considered at risk of infection). These guidelines also recommend that antimicrobials identified as critically important for humans not be used in food-producing animals for treatment or disease control unless susceptibility testing demonstrates the drug to be the only treatment option. Conclusions: To preserve the effectiveness of medically important antimicrobials, veterinarians, farmers, regulatory agencies, and all other stakeholders are urged to adopt these recommendations and work towards implementation of these guidelines.

The strategic plan for combating antimicrobial resistance in Gulf Cooperation Council States.

The Gulf Cooperation Council Center for Infection Control (GCC-IC) has placed the emergence of antimicrobial resistance (AMR) on the top of its agenda for the past four years. The board members have developed the initial draft for the GCC strategic plan for combating AMR in 2014. The strategic plan stems from the WHO mandate to combat AMR at all levels. The need for engaging a large number of stakeholders has prompted the GCC-IC to engage a wider core of professionals in finalizing the plan. A multi-disciplinary group of more than 40 experts were then identified. And a workshop was conducted in Riyadh January 2015 and included, for the first time, representation of relevant ministries and agencies as well as international experts in the field. Participants worked over a period of two and a half days in different groups. International experts shared the global experiences and challenges in addressing human, food, animal, and environmental aspects of controlling AMR. Participants were then divided into 4 groups each to address the human, animal, microbiological and diagnostic, or the environmental aspect of AMR. At the end of the workshop, the strategic plan was revised and endorsed by all participants. The GCC-IC board members then approved it as the strategic plan for AMR. The document produced here is the first GCC strategic plan addressing AMR, which shall be adopted by GCC countries to develop country-based plans and related key performance indicators (KPIs). It is now the role of each country to identify the body that will be accountable for implementing the plan at the country level.

Integron-associated antibiotic resistance in enteroaggregative and enteroinvasive Escherichia coli.

Ten enteroinvasive (EIEC) and 25 enteroaggregative (EaggEC) E. coli strains isolated from Senegalese patients were analyzed for their integron content. All strains were resistant to at least two antibiotics. Four EIEC and 15 EaggEC were found to carry a class 1 integron. An identical integron carrying a single dfrA5 cassette, conferring resistance to trimethoprim, was identified in all four EIEC strains. Five EaggEC strains harbored an integron with a single cassette, dfrA7, while the remaining 10 strains carried two integrons, one with a single cassette, aadA1a conferring resistance to streptomycin and spectinomycin, and the second one bearing two cassettes, dfrA13 and oxa5, the later being a beta-lactam resistance cassette. The presence of these integrons is worrying, because trimethoprim is largely used for diarrheal disease therapy in Africa. Thus, the presence of integrons in diarrheagenic strains is of public health importance because a limited number of antibiotics are available in developing countries.

High prevalence of trimethoprim-resistance cassettes in class 1 and 2 integrons in Senegalese Shigella spp isolates.

BACKGROUND: Integrons have a well-established role in the dissemination of resistance among Gram-negative pathogens and are thus a useful marker of antibiotic resistance. Shigellae are noteworthy for their multiple drug resistance, having gradually acquired resistance to most widely use and inexpensive antimicrobial drugs. METHODOLOGY: A total of 32 Shigella strains belonging to serotypes flexneri, dysenteriae, and boydii 20, a new Shigella serovar, resistant to at least four antibiotics were analyzed by molecular techniques. RESULTS: Class 1 integrons were the most prevalent (92.8%); class 2 integrons were found in 16 strains (57.1%). Fifty percent of the strains harboured both class 1 and 2 integrons (intI1 and intI2 genes); this combination of integrase genes was most prevalent in S. boydii 20 and S. dysenteriae strains. The class 1 integrons detected contained dfr and aadA cassettes, alone or in combination (dfrA5/dfrA15, or dfrA15-aadA1, dfrA1-aadA2), and an atypical cassette array with an insertion sequence (oxa30-aadA1-IS1). For class 2 integrons, we detected either the same cassettes as those found in Tn7 (dfrA1-sat1-aadA1-orfX) or truncated class 2 integrons without aadA1 or orfX. The tns genes were absent from all class 2 integrons.The distribution of integrons among RAPD profiles and serotypes revealed a clonal spread of integrons into serotypes and a transfer of integrons between different serotypes. CONCLUSIONS: The detection of integrons in a new Shigella serovar, in addition with a high integron prevalence among Shigella strains, confirms the propensity of shigellae to acquire and disseminate resistance determinants.