Confirmed E. coli isolates were subjected to molecular analysis investigating resistant mechanisms and genotypes with special emphasis on resistance determinants of clinical relevance and useful as markers in molecular epidemiology. Deliverables included data on the distribution of multi-drug resistant phenotypes and relevant resistance genes in the commensal microbiota of 3174 children aged 6-72 months from Bolivia and Peru. A widespread dissemination of MDR isolates and co-transferable resistance determinants could be described. In a random sample of 1.080 resistant E. coli isolates, 90% exhibited a multi-drug-resistance (MDR) phenotype. The two most common MDR phenotypes (ampicillin/tetracycline/trimethoprim-sulphamethoxazole and ampicillin/tetracycline/trimethoprim-sulphamethoxazole/chloramphenicol) could be transferred en bloc in conjugation experiments. The most common acquired resistance genes were: blaTEM, tet(A), tet(B), drfA8, sul1, sul2 and catI. Investigating the acquired resistance to sulphonamides, it was possible to describe for the first time in America the presence of sul3 gene. Moreover, 4 ceftriaxone-resistant E. coli isolates were collected, one from each urban area. These isolates were shown to produce CTX-M-type extended-spectrum-?-lactamases (ESBL): CTX-M-2 (the isolates from Camiri, Villa Montes and Moyobamba) and CTX-M-15 (the isolate from Yurimaguas). This was the first report of CTX-M-type enzymes in Bolivia and Peru, and also the first detection of CTX-M-15 in Latin America. Mechanisms of resistance spreading were complex and involved both clonal expansion of antimicrobial-resistant strains and horizontal transfer/recombination of mobile genetic elements harbouring resistance genes. Two years later the antimicrobial resistance screening has been repeated in the same study area enrolling 3193 healthy children in the same age cohort. The results revealed a notable trend toward the dissemination of CTX-M genes in commensal E. coli, which could represent an important reservoir of these emerging resistance determinants in the community. These findings underscore the magnitude of the problem of antimicrobial drug resistance in low-resource settings and the urgent need for surveillance and control of this phenomenon.
Evaluation and standardisation of a rapid screening direct-plating method for detection of antimicrobial resistance in commensal microbiota of the gut
Rapid screening methods, based on the detection of faecal resistant Escherichia coli strains directly on the McConkey plates, suffered from a lack of standardisation. With the aim to fill this gap, a study was carried out to evaluate the performance of a direct-plating method (DPM) for detection of antimicrobial resistance of faecal E. coli from healthy subjects, and to compare the results with those obtained with a conventional method. Faecal samples were obtained by rectal swabs or swabs from fresh stool from 71 healthy children aged 6-72 months from urban and rural areas of Peru and Bolivia. Microbiological analysis was performed contemporaneously by DPM and by conventional method. In the rapid screening method a faecal swab was directly plated onto McConkey agar plate and antimicrobial disks applied onto the seeded plate. The same faecal swab was also processed by standard agar disk diffusion method based on testing antimicrobial susceptibility in three randomly selected colonies from primary cultures of faecal samples on selective media. Raw data from rapid screening method were obtained by the direct reading of the plate and subjected to confirmatory analysis. A good concordance between the DPM and conventional method was observed in detecting carriage of resistant E. coli with a higher sensitivity of the DPM. The analysis of results allowed to define interpretive criteria for DPM raw data. Applying these criteria, DPM showed good sensitivity and specificity at very low cost (ten times cheaper than the conventional method). The DPM proved to be a very sensitive, specific, fast and cheap procedure to investigate the faecal carriage of drug-resistant E. coli and may represent an useful tool to conduct large-scale resistance surveillance studies and to monitor resistance-control programs cost-effectively, particularly in low resource countries.
Human antimicrobial use was investigated in two urban communities in Bolivia (Camiri and Villa Montes) and Peru (Yurimaguas and Moyobamba) in a total of 3174 households. The study population was represented by healthy children aged 6-72 months and their carers. Data on antimicrobial use at child, family and community levels were collected by questionnaire, while carers and providers conceptions regarding antimicrobial treatment of children were investigated by focus group discussions. Results indicated that the parents health seeking strategies include treatments with traditional medicines as well as an extensive utilisation of the public health care system. Often traditional medicine was used as first-choice treatment, regardless of the educational level of the parents. The majority of antimicrobials used to treat the children was obtained from the health care system, and was not given as self-medication. Drugs were provided to a high extent for pneumonia-like symptoms, but less frequently for cough/cold. Medical doctors showed little discrimination in antimicrobial prescription for illnesses recommended or not for antimicrobial treatment according to principles of the integrated management of childhood illnesses (IMCI) algorithm. As foreseen in the project, results contributed to the conceptual framework and design of the Information-Education-Communication (IEC)-Intervention campaign performed to control antimicrobial use and resistance in the given environment. The finalised HAUS methodology, applied in the post-intervention phase to a total of 3193 households of the same study area, is representing an usefull tool to evaluate the impact and expected improvement in health seeking behaviour attribuible to IEC intervention campaign. Analysis of data is still underway.
Development of an antimicrobial use and resistance monitoring system, applied to healthy children (aged 6-72 months) and their families in LIC
Human antimicrobial use was investigated in the study population through Household Antimicrobial Use Survey (HAUS), based on previously used and validated methods for collecting both quantitative and qualitative data. Quantitative data were collected performing Household interviews investigating carers of the selected study population by using a pre-tested questionnaire for assessing the families and the selected study population of children (aged 6-72 months) regarding issues of health seeking behaviour and consumption patterns of antimicrobials including self-medication. Qualitative data were collected by Focus group discussions with family representatives and local health care providers investigating their conceptions regarding views and ideas around antimicrobial use, self-medication, and community health care system. Antimicrobial resistance screening (ARS) was performed on bacterial isolates from the commensal microbiota of the gut (Escherichia coli). Samples of the gut microbiota were obtained by rectal swabs or swabs from fresh stool from the youngest recruitable child in the target age cohort. The antimicrobial susceptibility of E. coli was analyzed by means of a validated and standardised rapid resistance screening method. Briefly, each swab was used to inoculate a plate of MacConkey agar and antimicrobial-containing discs were then applied onto the medium surface. The antimicrobials tested are including ampicillin, ceftriaxone, teytracycline, trimethoprim-sulphamethoxazole, chloramphenicol, streptomycin, kanamycin, gentamicin, amikacin, nalidixic acid and ciprofloxacin. Results from MacConkey agar plates were recorded after a 18 h-incubation at 37°C. Colonies with a morphology resembling that of E. coli grown inside the zones of inhibition that define susceptibility to each antibiotic, were confirmed at species level and scored as resistant isolates. The data collected on pre-printed HAUS and ARS forms were electronically stored and validated as foreseen in the Epi-Info 2000 programme. Surveillance of resistance is not itself an intervention that will contain resistance, but which is of critical importance both to provide information on the magnitude and trends in resistance and to monitor the effect of interventions performed to control antimicrobial use and resistance. The developed monitoring system ARS and HAUS including the rapid resistance screening method, will be proposed to the national regulatory authorities for continuous or interval monitoring subsequent to this project.
Development and validation of molecular tools for the genetic analysis of resistance mechanisms and genotypes
- Detection of resistance genes and integrons. Molecular methods, based on DNA hybridization or PCR and direct sequencing, for detection of the following resistance genes were developed and validated: -lactamases resistance genes (blaTEM, blaSHV, blaCTX-M, blaPER and various blaOXA), tetracycline resistance genes (tet(A), tet(B), tet(C) and tet(D)), phenicols resistance genes (catI and cmlA), trimethoprim resistance genes (dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA13, dfrA14 and dfrA17), sulphonamides resistance genes (sul1, sul2 and sul3). As in gram negatives the aminoglycoside resistance determinants are known to be usually integron-associated, a PCR mapping approach was developed, suitable for identification of integron-borne aminoglycoside resistance genes. The method is based on PCR amplification of integron cassette arrays using primers designed on conserved integron regions (class 1 and class 2 integrons), possibly in combination with primers specific for the various aminoglycoside resistance genes. Characterization of the resistance genes is then carried out by direct sequencing. For this purpose primers for amplification or sequencing of the most representative aminoglycoside resistance genes were designed. - Plasmid replicon typing. A methodology for the characterization of plasmid replicon types, as marker of plasmid backbone, was implemented to better understand the mechanisms of resistance spreading in the E. coli isolates from the ARS studies. This methodology is based on a multiplex-PCR (Carattoli et al., J Microbiol Methods 2005) and/or a DNA hybridization approach (Couturier et al., Microbiol Rev 1988). Several experiments were carried out to standardize the methodology in our laboratory, using a collection of plasmids harbouring known replicon types. - Genotyping. A genotyping methodology was implemented to investigate the clonal relationships among the resistant E. coli isolates. This methodology is based on PCR amplification using three different decamer primers (Random Amplification of Polymorphic DNA, RAPD) (Pacheco et al., J Clin Microbiol 1997), and on a phylogenetic analysis carried out with the Diversity Database program (Bio-Rad Laboratories, Hercules, California) to compare different band patterns. Several experiments were carried out to standardize the methodology in our laboratory, using a collection of E. coli clinical isolates of different origins.
Development of a conceptual framework to control antimicrobial use and resistance in low-income countries
A conceptual framework was developed to describe antimicrobial use and resistance in healthy children aged 6-72 months from urban and remote rural communities of Bolivia and Peru. Antimicrobial use was determined by the household survey methodology and antimicrobial resistance in commensal organisms of the gut (Escherichia coli) by using a simple and robust screening method. Selected resistant bacteria were further investigated at phenotype and genotype level. Other factors influencing the phenomenon were considered, including national drug policy, non-human use of antimicrobials and water contamination. To improve the situation a community intervention package focusing on rational use of antimicrobials was evaluated. It was developed and implemented in collaboration with the local public and private health services on the basis of the generated results enforced by the national drug policy. The framework aimed to contribute to a more appropriate antimicrobial use and evidence based antimicrobial policy.
Antimicrobial resistance was analysed in the commensal microbiota of the gut (Escherichia coli) in healthy children aged 6-72 months by a rapid resistance screening method used on stool specimens and confirmed by a standard antimicrobial susceptibility test. A total of 3174 children in the target age cohort were enrolled from four urban communities in Bolivia and Peru. Moreover, antimicrobial use and bacterial microbial resistance were investigated in 93 inhabitants of Angaiza, a remote community of the Peruvian Amazonas characterised by very low human use of antimicrobials. The adopted screening method was refined and confirmed to be particularly suitable to monitor resistance-control programs cost-effectively in low resource countries. Results showed a high prevalence (70%-95%) of healthy carriers of E. coli resistant to a number of older antimicrobial agent (ampicillin, tetracycline, trimethoprim-sulphamethoxazole, chloramphenicol). A relatively high prevalence (18%-30%) of carriers of E. coli resistant to other agents (kanamycin, gentamicin, nalidixic acid and ciprofloxacin) was also observed, while the carriage of E. coli resistant to expanded-spectrum cephalosporins and amikacin was uncommon (<0.5%). Milestones consisted in the collection of 18.042 E. coli isolates from which 2000 representatives were selected and scored to undergo further phenotypic and genotypic analysis. In the isolated community in Peru, where antimicrobial use was very limited, unexpectedly high resistance rates were detected suggesting that, in some cases, the spread and maintenance of resistant strains could be not directly related to antimicrobial consumption. The antimicrobial resistance screening was repeated in the same study area in 3193 healthy children aged 6-72 months. The investigation was carried out after the implementation of an Information-Education-Communication Intervention campaign performed to control the rational use of antimicronbials in the community. As the time period passed between the intervention and the microbiological study was too small, the antimicrobial resistance screening, did not represent a valuable indicator to evaluate the impact of the Information-Education-Communication campaign. Results, besides confirming the high resistance rates, showed an alarming significant increase in the resistance rates to quinolones (57% vs. 35% for nalidixic acid and 33% vs. 18% for ciprofloxacin) and ceftriaxone (1.7% vs. 0.1 %). The trend was similar overall in each of the 4 studied areas.
Development of a simple and inexpensive phenotypic test for rapid Escherichia coli identification during fieldwork
Presumptive identification of E. coli isolates based on the colony morphology on McConkey agar can be affected by poor specificity, especially when carried out by inexperienced personnel. Moreover, approximately 10% of E. coli isolates are lactose-negative, and these isolates are going to be missed if only lactose-positive colonies are considered. To facilitate the process of selection of E. coli isolates grown onto the screening plates, a simple and inexpensive phenotypic test for rapid identification of E. coli, suitable for field work, was developed. The test is based on the detection of the enzyme Beta-D-glucuronidase using the substrate 4-methylumbelliferyl-Beta-D-glucuronide (MUG) which, upon hydrolysis by the enzyme, yields the fluorogenic compound 4-methylumbelliferone. Production of this enzyme is considered to be diagnostic for E. coli: 94 to 97% of E. coli strains are reported to produce this enzyme, while other bacteria that produce this enzyme do not grow on media selective for coliforms (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354). The test is carried out as follows: 10 microL of MUG 80 microg/mL, dissolved in 0.05 M Sorensen s phosphate buffer (pH 7.5), is dispensed in a well of a microtiter plate. A small amount of the colony to be tested is taken with a sterile toothpick and suspended in the MUG solution. After 2 hours of incubation at 37°C the plate is observed under UV light (a portable battery-operated Wood�s lamp is suitable). An intense blue fluorescence indicates a positive reaction, while no or a weak fluorescence indicates a negative reaction. In each test positive and negative controls are always inserted as references. The test was validated using 250 isolates from the pilot study. It allowed correct identification of 91% of E. coli isolates (in agreement with the data from the literature that report that Beta-D-glucuronidase production occurs in approximately 90% of E. coli isolates - McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354), with no false positives. Compared to the MUG-tests based on the use of culture media supplemented with MUG, this test is considerably less expensive (the cost is approximately 0.002 Euro per test) and is feasible for routine application in fieldwork in low-resources settings such as those participating in this project. Moreover, the use of microtiter plates and small volumes facilitates a quick and simple analysis of a high number of strains. The major usefulness of a similar test, which allows rapid, cheap and specific confirmation of E. coli isolates, consists in reducing the rate of false positives derived from collection of lactose fermenters. Moreover, since production of Beta-D-glucuronidase is not related to production of Beta-galactosidase, the test allows reliable identification of E. coli isolates among lactose nonfermenting colonies, thus avoiding the systematic missing of similar isolates if lactose-positivity is considered for screening purposes. Due to these features, it was decided to use this test during the ARS-Baseline activities in Bolivia and Peru, as a supporting tool for interpretation of results of the rapid screening method. During the first days of the ARS-Baseline activities, the MUG-test was carried out for several coliform and atypical colonies, and this approach allowed to considerably improve the ability of the local personnel to classify isolates grown on MacConkey agar plates. Although this training was rather time-consuming, due to the necessity of a two-step reading of plates (selection of the colonies to test with MUG, followed by observation of the colonies knowing the results of the MUG-test), it resulted very helpful to increase the ability of the local personnel to differentiate E. coli colonies from the usual mucoid, lactose-fermenting, MUG-negative colonies of Klebsiella spp.. The MUG-test was also used to recognise the morphology of lactose-nonfermenting E. coli colonies. After a first period of training, the MUG-test was used whenever doubts persisted.
Study on factors influencing the kinetics of antimicrobial use and antimicrobial resistance in the community
The interrelationship between human antimicrobial use and antimicrobial resistance was analysed taking into account influencing factors, including current pharmaceutical regulatory framework, quality of antimicrobials, agricultural and veterinary use of antimicrobials and bacterial contamination in water sources. In Bolivia, the national regulatory system was analysed by means of significant quality indicators. Based on the results, a draft to support an updated pharmaceutical regulatory framework was elaborated and is underway to be proposed to the government. The document is entitled "National drug policy, pharmaceutical regulatory framework and rational use of antimicrobials". Moreover, the level of water contamination was assessed in a small field investigation performed in the study area. In Peru, a desk study to assess veterinary use of antimicrobials has been carried out in Iquitos (Departamento de Loreto), reporting information available in 2002 at the local University of Veterinary and the US Food and Drug Administration. The problems detected were disseminated and discussed during the educational meetings. Results included both the increased awareness regarding current drug regulations and knowledge on the high level of antimicrobials used for animal breeding and the bacterial contamination in water sources.
The dissemination of outcomes included a a major international meeting and dissemination seminars, at the presence of research collaborators, representatives of the key actors and organisations involved in health system research, drug policy making and health sector reforms, with the objective of debating the results. The project methodologies were presented including the intervention strategy, the developed antimicrobial use and resistance monitoring system (HAUS and ARS and the standardised rapid antimicrobial resistance screening method to support governmental institutions involved in public health and drug policy making. Data on antimicrobial and gender specific use among 6-72 month old children and household memebers and carers and providers conceptions regarding antimicrobial treatment of children were reported in peer reviewed scientific journals (4 articles), presented in national and international conferences (10 presentations) and diffused by mass media. Further preparation of manuscripts is underway. Translation in Spanish of a significant paper was performed to increase locally the diffusion of results. Moreover, two Medical students, two MDs post-graduating in infectious diseases and two PhD students promoted through the project. Progress reports and final report were submitted to the European Commission as requested.
In consideration that the commercial biochemical identification systems for E. coli are expensive and time-consuming, and therefore poorly suitable for analysing a large number of samples to be investigated in monitoring screening, a method for rapid, inexpensive and reliable identification of E. coli by means of filter DNA hybridisation was developed. Two different targets were selected to develop species-specific DNA probes for E. coli: the lacZ gene, encoding galactosidase, and the uidA gene, encoding glucuronidase, which has already been used to develop species-specific DNA probes for E. coli (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354). The hybridization probe for the lacZ gene was generated by PCR amplification of a 340-bp segment of the lacZ gene from E. coli CCUG24T, using primers Bgal-fwd (5 -TCTGGAAGATCAGGAATATGTGG) and Bgal-rev (5 -ATAGAGATTCGGGATTTCGGC). The hybridization probe for the uidA gene was generated by PCR amplification of an 882-bp segment of the uidA gene from E. coli CCUG24T, using primers uidA-fwd (5-TGGGCATTCAGTCTGGATC) and uidA-rev (5 -GCACCATCAGCACGTTATC). The specificity of each probe was tested in a set of preliminary colony blot experiments using reference strains of E. coli, Klebsiella spp., Serratia spp., Citrobacter spp., Shigella spp., Providencia spp., Kluyvera spp. and Salmonella enterica. In these experiments the uidA probe was found to be more specific that the lacZ probe, and for this reason it was selected for further development. To validate the uidA probe, it was tested on a sub sample of 466 isolates collected during the pilot study, and results, obtained in a blinded fashion, were compared with those obtained with the conventional API 20E biochemical identification system. The two methods were in agreement with all the isolates, with the exception of 3 isolates which were identified as E. coli by the API 20E system but were not recognized by the uidA probe, and of 2 isolates which gave a positive hybridization signal with the uidA probe but couldn�t be identified with certainty with the API20E system. These results are overall consistent with previous observations indicating that 97 to 100% of E. coli strains carry the uidA gene (McDaniels et al., Appl. Environ. Microbiol., 1996, 62:3350-3354). According to these results, the colony blot protocol based on the uidA probe appeared to be a sensitive and specific method for identification of E. coli isolates. In comparison with conventional biochemical testing, this method is less time-consuming and much cheaper (less than 1 Euro vs. 10 Euro per tested isolate). For these reasons, it was decided to adopt this method for E. coli identification in the baseline ARS study.
Exportable Intervention package strategy to improve rational use of antimicrobials in low-income countries
Decreased antimicrobial selective pressure is assumed to decrease the prevalence of resistant strains. With the aim to reduce antimicrobial consumption, a multi-faceted intervention campaign was carried out including the education of providers (widely accepted recommendations for good clinical diagnosis and treatment) and measures to improve the public�s knowledge about the risks and benefits of antimicrobial therapy. The Information-Education-Communication (IEC) package was designed and finalised according to problems of rational use and resistance detected by means of household surveys, antimicrobial resistance screening, genotypic analysis and influencing factors during the pre-intervention phase. The multi-faceted activities were realized, by considering a three step program with Academic detailing, to present and discuss the identified problems among providers (medical doctors, pharmacists) with the aim of improving antimicrobial dispensing; Peer network, established by research team members and interested providers, to develop strategies for improving the rational use of antimicrobials in the community and Community intervention, to bring up changes in attitudes and habits on antimicrobial use and self-medication. The activities were performed in sequence for a minimum of 18 months in the intervention cities from both Bolivia and Peru. A multifaceted intervention campaign was realized addressing journalists, teachers, students, mothers and families using marionettes, theatre pieces, puppets, rotafolio, radio spots and mass media. Posters and panels were distributed in the cities; messages focused on self-medication and compliance to treatment. In Peru, all preparatory and executory activities undetaken to realise the Information-Education-Communication Intervention were summarised in a brochure entitled "Proyecto ANTRES - Intervención en la población de Yurimaguas", that is underway to be presented to the Ministry of Health.