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A general method for rapid determination of antibiotic susceptibility and species in bacterial infections

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To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have
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   1 A General Method to Rapidly Determine Antibiotic Susceptibility and 1 Species in Bacterial Infections 2 3 Running title: Rapid Antibiotic Susceptibility Testing 4 5  Anja Mezger  †   , Erik Gullberg   ‡  , Jenny Göransson  §   , Anna Zorzet   ‡1  , David Herthnek  †   , Eva 6 Tano ˣ   , Mats Nilsson †  * 2  , Dan I. Andersson  ‡ * 3   7 8 † Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm 9 University, Box 1031, 17121 Solna, Sweden 10 ‡ Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden 11 §  Q-linea AB, Uppsala, Sweden 12 ˣ Department of Medical Sciences/Section of Clinical Bacteriology, Uppsala University, 13 Sweden 14 * Corresponding   authors, equal contribution 15 16 Word count for the abstract: 155 17 Word count for the text: 4132   18 1   Department of Medical Sciences/ReAct, Uppsala University, Sweden 2  Mats.Nilsson@scilifelab.se   3  Dan.Andersson@imbim.uu.se   JCM Accepts, published online ahead of print on 19 November 2014J. Clin. Microbiol. doi:10.1128/JCM.02434-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.   2 Abstract 19 To assure correct antibiotic treatment and reduce unnecessary use of antibiotics there is 20 an urgent need for new rapid methods for species identification and determination of 21 antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general 22 method to rapidly identify the bacterial species causing an infection and determine their 23 antibiotic susceptibility profiles. An initial short cultivation step in the absence and 24  presence of different antibiotics was combined with a sensitive species-specific padlock 25  probe detection of the bacterial target DNA to allow determination of growth (i.e. 26 resistance) and no growth (i.e. susceptibility). A proof-of-concept was established for 27 urinary tract infections where we applied the method to determine the antibiotic 28 susceptibility profile of  E. coli for two drugs with 100% accuracy in 3.5 hours. The short 29 assay time from sample to readout enables fast appropriate treatment with effective 30 drugs, and minimizes the need of prescribing broad-spectrum antibiotics due to unknown 31 resistance profiles of the treated infection. 32 33 Introduction   34 Over-prescription and extensive use of antibiotics have selected for resistant bacteria at 35 an alarmingly rapid rate and we are now facing one of the greatest medical challenges of 36 our time (1). Today, both diagnosis of bacterial infections and determination of antibiotic 37 susceptibility profiles (ASP) are slow and tedious processes. As a consequence, the 38  patient might be given an antibiotic that has no effect in the case of infection with 39 resistant bacteria. Thus, there is a considerable need for new techniques enabling quick 40 and specific diagnosis along with characterization of an ASP in order to guide correct 41   3 treatment, reduce the use of broad-spectrum antibiotics and slow down resistance 42 development. 43 The last few decades we have seen an amazing development of novel molecular 44 methods to detect bacterial pathogens and their resistance genes and resistance mutations 45 (2-5). These new hybridization/PCR-based methods are generally faster and more 46 sensitive than the classical phenotypic methods, but they also suffer serious drawbacks 47 that have often reduced their general use. An intrinsic limitation of all genotypic methods 48 that identify resistance mutations or genes is that they only detect the potential for 49 resistance (i.e. presence of a resistance gene/mutation), whereas phenotypic methods 50 detect realization of susceptibility (i.e. no growth in presence of antibiotic). For a 51 clinician, the latter measure is far more relevant as a basis for a therapeutic decision. 52 Padlock probes are oligonucleotides with target specific ends, which upon perfect 53 target recognition can be enzymatically joined (8). Reacted probes can be amplified by 54 rolling circle amplification (RCA). RCA is a linear amplification technique for 55 replication of DNA circles, such as reacted padlock probes, and the product is a single 56 stranded DNA concatemer containing around 1,000 copies of a 100-mer template circle 57 after one hour of replication (9, 10). After monomerization by restriction enzymes, these 58  products can be religated and further amplified by a second round of RCA (11). The 59 amplification products are typically labeled with fluorescence and detected by 60 fluorescence microscopy or with a scanner (12, 13). 61 In order to develop a general and quick method for both diagnosis and generation 62 of a corresponding ASP, we merged the classical growth-based phenotypic test with a 63 highly sensitive and specific genotypic test to examine the resistance profile in a bacterial 64   4 sample. A sample is cultivated for a short period in the absence and presence of different 65 antibiotic substances. To detect growth or no-growth, the sample DNA is harvested and 66 exposed to padlock probes designed to detect target sequences positioned in the 16S 67 rRNA region. Reacted probes are amplified by RCA, and RCA products are counted in a 68 high-performance fluorescence detector, providing precise digital quantification to 69 sensitively detect differences in DNA copy number. An increase in DNA amount in 70  presence of the tested drug shows that the bacteria are phenotypically resistant and, 71 conversely, no growth in presence of drug indicates susceptibility. We demonstrate the 72 specificity of each padlock probe to its respective target, and we illustrate the 73  performance of the method by determining the ASP for  E. coli  which is the most 74 common infectious agent in urinary tract infection (UTI) in 88 clinical samples in one 75 retrospective and one prospective study. The time from urine sample to readout is 3.5 76 hours and therefore, this method has a significant time advantage compared to the 77 commonly used culture-based methods. 78 79 Methods 80 Rationale for the assay. The rationale for the assay is schematically outlined in Fig. 1 81 and it includes four central steps: (i) growth in absence and presence of different relevant 82 antibiotics, (ii) total DNA preparation, (iii) species-specific padlock probe ligation and 83 amplification, and (iv) digital readout of amplified reacted probes to assess any change in 84 the quantity of rDNA in absence and presence of antibiotics. 85 86 The reaction chain begins with a short (30 min to two hours) cultivation of the urine 87   5 sample supplemented with LB (Fig. 1A). This step distinguishes a susceptible strain from 88 a resistant strain as only bacteria with a resistant phenotype can grow in the presence of 89 the antibiotic, and thereby generate more genomes (rDNA). Genomic DNA was extracted 90 from the cells by using NaOH and heat treatment to lyse cells. Heat treatment also served 91 to randomly fragment and denature the genome prior to padlock probe hybridization (Fig 92 1B). This crude cell lysate in urine/LB was used as input for the molecular assay without 93 the need for purification. 94 95 Padlock probes were designed to target species-specific sequences in the 16S RNA gene 96 of  E. coli ,  P. aeruginosa  and  P. mirabilis  (Table 1). To enrich for the target sequences, 97 cell lysates were mixed with complementary biotinylated capture oligonuleotides and 98 streptavidin-coated magnetic beads (Fig. 1C). These served as a solid support and 99 allowed exchange of the urine/LB with the ligation mixture containing the padlock 100  probes. Hybridized and ligated padlock probes were amplified by RCA (Fig. 1D-E) and 101 digested by restriction enzymes (Fig. 1F). The monomers were re-ligated for a second 102 round of RCA to further increase the number of RCPs. Two oligonucleotides 103 complementary to the RCA products and labeled with 5’- Cy3 were hybridized to the 104 RCP during the second round of amplification (Fig. 1G). A commercially available high- 105  performance fluorescence detector (Aquila 400, Q-linea AB, Sweden) was used, since it 106 allows for a fast and precise digital read-out with a high dynamic range and quantitative 107  precision. If necessary, samples were diluted prior to measuring. 108 109 Processing of urine samples. We analyzed 88 urine samples from patients with UTI 110
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