The aim of the present research proposal is to compare the molecular characteristics of ESC-R Ec of human and veterinary origin collected in Switzerland. To do this, we will analyze Ec isolates responsible for intestinal colonization or infection in the above different hosts. Performing such investigation in this unique small area may help to understand the dynamic of emergence of ESC-R Ec and understand the importance of the animal reservoirs in the initial spreading of ESBL/pAmpC-Ec among humans. Origin, number, and model approaches implemented to collect Ec isolates
A large set of ESC-R Ec (i.e., at least 200 isolates) recently collected from humans, farm animals and pets will be analyzed. In particular, the following collections of ESBL/pAmpCs-Ec isolates are already available and under initial characterization (see “preliminary results” section):
1. Thirty-four ESBL- and pAmpC-producing Ec isolates (n=22 and n=12, respectively) recovered from cloacal swabs of broilers (n=30), swine (n=2), and cattle (n=2). These strains were obtained analyzing an overall number of 600 cloacal samples collected by ZOBA (Institute of Veterinary Bacteriology, University of Bern) during October 2010 to April 2011 and involving 5 slaughterhouses located in 14 different Cantons. Further 26 ESC-R Ec isolates were identified in the same animal species during May to August 2011. See below “preliminary results” section for the first group of 34 ESC-R Ec isolates.
Model.
Representative samples were taken according to the guidelines of the Swiss National Monitoring Program on Antimicrobial Resistance in Food Animals [20]. The sampling strategy consists of collecting from each slaughterhouse a number of samples that is proportional to the number of animals slaughtered at each establishment per year. The sampling is also evenly distributed across each month of the year. The samples were randomly collected at the 5 biggest broiler abattoirs and the 14 biggest pig and cattle abattoirs where over 80% of livestock in Switzerland are slaughtered. Only one sample was taken per animal holding for pigs and cattle, and one pool of 5 animals per holding was analyzed for broilers.
2. Seventy-two ESBL-Ec isolates (inpatients and outpatients) collected from infected or colonized patients (including 11 children) recover by the Institut für Infektionskrankheiten (IFIK, University of Bern) at the Inselspital during May 2008 to September 2009. Furthermore, ESBL-Ec isolates collected in the faeces of the household contacts (i.e., relatives) of the above discharged 72 patients were isolated. See below “preliminary results” section.
Model.
This study was an observational prospective surveillance conducted at the University Hospital of Bern (Inselspital, Switzerland). The analyzed population included pediatric and adult patients hospitalized or referring to Inselspital during May 1st 2008 to September 30th 2009 and presenting with a newly detected clinical sample with an ESBL-positive isolate.
An index patient was defined as an in- or outpatient with a newly recognized infection or colonization with ESBL-Ec isolates. Hospital contact patients were defined as hospital room mates who shared the same wardroom, intensive or immediate care room for ³ 48 hours with index patients. Household contact persons were defined as persons that shared the same household with the index patient on a regular basis. Transmission was defined as having identical blaESBL genes and a clonally-related ESBL-Ec isolate.
During this study, the maximum follow up period for in- and outpatients was 12 months. For index inpatients, samples were obtained at time of first detection of the ESBL-producing organism and weekly thereafter until in hospital death or discharge. In case of the latter, additional samples from the household contact were received trimonthly thereafter. Screening included stools but also other clinical samples. For index outpatients, exclusively stool samples were obtained at time of first detection of the ESBL-producing organisms and trimonthly thereafter (or with a new hospitalization at our center if any). Hospital contact patients were screened weekly until one week after physical separation from the index patient and had another screening at hospital discharge if the last screening was performed >7 days before discharge. For household contact persons stool samples were obtained every 3 months, until both the index and contact showed negative screening results. Screening of hospital and household contacts stopped at latest 1 year after the recruitment of the corresponding index patient (i.e., latest on September 30th 2010).
Stool samples were analyzed with different ESBL selective culture media: ChromID ESBL agar (BiomérieuxÒ), BLSE agar (AESÒ), a bi-plate with 2 selective media (MacConkey agar plus ceftazidime and Drigalski agar plus cefotaxime at a concentration of 2 and 1.5 mg/L, respectively) and CHROMagar ESBL (chromagarÒ). Growing colonies (i.e., third-generation cephalosporins resistant and suspicious for ESBL production) were first subject to species identification by using standard biochemical methods and the Vitek2 system (bioMérieux SA).
An exposure network graph was created using Pajek, Program for Large Network Analysis [http://vlado.fmf.uni-lj.si/pub/networks/pajek/]. Statistical analysis was performed using STATA 10 (Stata Corporation).
3. Twenty-six ESC-R Ec isolates collected by Dr. André Burnens (Medica, Medizinische Laboratorien Dr. F. Kaeppeli AG, Zurich) from the urines (n=21) and respiratory secretions (n=5) of patients hospitalized in long-term care facilities (LTCFs). See below “preliminary results” section.
4. Fourty ESC-R Ec isolates obtained from pets (of which 13 from dogs) and horses (n=16) during 2008-2010. This isolates have been routinely identified by ZOBA. Clinical data regarding the horses and pets are available and can be analyzed as recently done for infections due to MDR Acinetobacter baumannii isolates (reference). See below “preliminary results” section.
5. Thirty pan-sensible Ec isolates responsible for bloodstream infection in patients recovered at the Inselspital during 2009-2011 (10 strains each year) and 30 additional pan-sensible isolates responsible for urinary tract infection in outpatients referring to the Laboratory of Clinical Microbiology of Inselspital (IFIK) during 2011. These isolates will be used as negative controls, analyzing with particular emphasis their clonality (e.g., rep-PCR and MLST).
Along with the above large collection of Ec isolates, additional strains that will be collected during the ongoing Resistance Monitoring at ZOBA will be provided and included in the project. Furthermore, additional human isolates will be provided by Drs. Endimiani and Muhlemann (IFIK) and by Dr. André Burnens (Medica). In particular, the IFIK is now focusing on ESC-R Ec (both ESBL and pAmpC producers) isolates responsible for infection and or colonization in pediatric patients, whereas Medica is collecting numerous isolates from elderly patients admitted to nursery homes and LTCFs. These are two settings that have been rarely explored for the presence of ESBL/pAmpC producers.
A recent study showed that people working at meat-processing factories in Switzerland harbored ESBL genes related to those found in animals [41]. This category of person may play an important role in the distribution of resistance as in has been demonstrated with associated MRSA. We will ask permission to Institute of Food safety, University of Zurich, for providing us some of these strains for molecular characterization.
Objective #1. Characterization of resistance genes. What are the molecular mechanisms responsible for β-lactam, quinolone and aminoglycoside resistance in ESC-R Ec detected in Switzerland? Are there differences in the molecular background of resistance between human and animal isolates? Are the antibiotic resistance genes spreading in Switzerland the same to those reported in other EU countries?
PCR/DNA sequencing will be used to characterize resistance genes. As mentioned, resistance to ESC in Ec is usually due to expression of ESBLs and/or pAmpCs [8, 9]. Quinolones resistance can be due to chromosomal mutations in the QRDR of gyrA and gyrB and/or expression of plasmid-mediated quinolone resistance (PMQR) determinants (various qnr, aac(6’)-Ib-cr, and qepA genes) [26, 60]. Aminoglycosides resistance is due to enzymatic inactivation mediated by AMEs and/or enzymatic methylation of nucleotides within the 16S rRNA (i.e., plasmid-mediated 16S rRNA methylases) [33, 34].
Objective #2. Analysis of clonality. Are the ESC-R Ec responsible for infection/colonization in humans and animals genetically related? Are the non-ESC-R isolates genetically related to those carrying blaESBL/pAmpC genes? Are the Ec clones spreading in Switzerland comparable to those reported in Europe? Is the “hyper-epidemic” CTX-M-15-producing Ec clone ST131 present in Swiss humans and/or animals?
The Multilocus Sequencing Typing (MLST) and the PFGE are considered the “gold standards” for studying clonality of bacterial strains [61]. However, the Repetitive-Extragenic Palindromic PCR (rep-PCR) is a more rapid alternative technique that uses primers that target repetitive sequences scattered throughout bacterial chromosome. The amplified DNA fragments, when separated by electrophoresis, constitute a genomic fingerprint [62-66].
Objective #3. Phylogenetic groups, pathotyping and serotyping. Are the virulence factors of ESC-R Ec of humans and animals origin the same? Are there resistance backgrounds specifically related to pathotype, serotype, or phylogenetic types?
Phylogenetic groups of ESC-R Ec isolates will be established using PCR, whereas pathotypes and serotypes will be defined using a new microarray platform that we recently designed.
Objective #4. Characterization of plasmids. Are the plasmids present in ESC-R Ec of human and animal origin genetically similar? What are the main Inc groups of plasmids spreading in ESC-R Ec detected in Switzerland? Are these Inc groups common to those reported in animals and humans in other EU countries?
PCR-based replicon typing (PBRT), plasmid-MLST (pMLST) and restriction fragment length polymorphism (RFLP) will be used to study and classify plasmids [55, 67, 68]. The whole genome sequence of four representative plasmids will be obtained using 454-pyrosequencing technology [69, 70].
