Lability of the pAA Virulence Plasmid in <i>Escherichia coli</i> O104:H4: Implications for Virulence in Humans

<div><p>Background</p><p><i>Escherichia coli</i> O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) <i>E. coli</i>. The strain displays “stacked-brick” aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.</p><p>Methodology/Principal Findings</p><p><i>E. coli</i> O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS) were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1%) patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (<i>P</i>≤0.01). This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (<i>P</i>  = 0.001).</p><p>Conclusions/Significance</p><p>The pAA plasmid can be lost by <i>E. coli</i> O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.</p></div

pAA is lost by the outbreak strain in the course of infection.

<p>Enrichment cultures of the initial and follow-up stool samples from patients A to G diluted to give rise to 150–200 well-separated colonies were plated on ESBL agar and pAA-positive colonies were identified using colony hybridization with the pCVD432 probe. The percentages of pCVD432-positive colonies among all colonies grown on the plates were calculated. The numbers above the columns indicate the time interval (days) between collection of the initial (black bars) and follow-up (white bars) stool samples. In all cases the percentage of pAA-positive colonies in the follow-up stool is significantly lower than that in the initial stool (<i>P</i>≤0.01; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone-0066717-t002" target="_blank">Table 2</a>).</p

Loss of pAA by EHEC O104:H4 outbreak strain as demonstrated by plasmid profiling.

<p>Isolated plasmids were separated using 0.6% agarose gel, visualized by staining with Midori green and photographed. Lane M, molecular size marker (plasmids from <i>E. coli</i> 39r861). In lanes 1 to 10, plasmid profiles of EHEC O104:H4 outbreak isolates from the following patients are shown (patients’ designations refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone-0066717-t001" target="_blank">Table 1</a>): Lane 1, initial isolate from patient A (pAA-positive); lane 2, follow-up isolate from patient A (pAA-negative); lane 3, initial isolate from patient C (pAA-positive); lane 4, follow-up isolate from patient C (pAA-negative); lane 5, initial isolate from patient G (pAA-positive); lane 6, follow-up isolate from patient G (pAA-negative); lane 7, reference EHEC O104:H4 outbreak isolate LB226692 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Bielaszewska1" target="_blank">[8]</a>; lane 8, initial isolate from patient H (pAA-negative); lane 9, follow-up isolate from patient H (pAA-negative). Sizes of the pAA (75 kb) and pESBL (88 kb) plasmids are indicated on the right side.</p

Characteristics of pAA-positive and pAA-negative EHEC O104:H4 isolates^a and clinical outcomes of infection in the respective patients.

a<p>The identity of all isolates as EHEC O104:H4 outbreak strain was confirmed using the multiplex real-time PCR targeting <i>rfb</i>_O104, <i>fliC</i>_H4, and <i>stx</i>_2a<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Zhang1" target="_blank">[24]</a> and multilocus sequence typing, which demonstrated that all belong to ST678 typical for the outbreak strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Bielaszewska1" target="_blank">[8]</a>.</p>b<p>HUS, hemolytic uremic syndrome; BD, bloody diarrhea; D, diarrhea without visible blood.</p>c<p>1, initial isolate; 2, follow-up isolate; the number in parenthesis indicates the time interval between recovery of the initial and the follow-up isolate.</p>d<p>+/+, PCR amplicon of corresponding size and hybridization signal on the 75-kb plasmid present; −/−, no PCR amplicon, no hybridization signal present.</p>e<p>Sizes of plasmids in kilobase pairs (kb).</p>f<p>Stx2a titers were defined as the highest dilutions of sterile culture filtrates that caused cytotoxicity in 50% Vero cells after 72 h.</p>g<p>AA, aggregative adherence pattern (HCT-8 cells).</p

Qualitative and quantitative analyses of pAA-positive bacteria in the paired initial and follow-up stool samples.

a<p>HUS, hemolytic uremic syndrome; BD, bloody diarrhea; D, diarrhea without visible blood.</p>b<p>1, initial stool sample; 2, follow-up stool sample; the number in parenthesis indicates the time interval between collection of the initial and the follow-up stool sample.</p>c<p>+, the isolate contained all pAA-encoded virulence genes (<i>aatA</i>, <i>aggR</i>, <i>aggC</i>, <i>aap</i>, <i>sepA</i>) in PCR and harbored a 75-kb plasmid hybridizing with pCVD432, <i>aggR</i>, <i>aggC</i>, <i>aap</i>, and <i>sepA</i> probes. -, the isolate lacked all pAA-encoded virulence genes in PCR and lacked the 75-kb plasmid in plasmid profiling.</p>d<p>+, an amplicon of corresponding size was obtained from the whole stool culture harvested from ESBL agar in PCR with primers pCVD432/start and pCVD432/stop <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066717#pone.0066717-Schmidt1" target="_blank">[30]</a>;</p><p>-, no PCR amplicon from the whole stool culture was obtained with these primers.</p>e<p>Determined by colony blot hybridization of stool cultures plated on ESBL agar with the pCVD432 probe and calculated from the total numbers of colonies grown on the plates.</p>f<p>Paired Student's <i>t</i> test (<i>P</i><0.05 considered significant); n.a., not applicable.</p

Additional file 1: of Genome-based analysis of Carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008-2014

Figure S1. Origin of the 107 German carbapenemase-producing K. pneumoniae isolates. Regions are shown, where isolates originated from. Isolates from Saxony could not be elucidated further due to the lack of additional geographic information. Number of isolates is given by the size of the circle (see legend). Image is from: © Bundesamt für Kartographie und Geodäsie, Frankfurt am Main, Germany. Figure S2. Virulence gene content in 107 carbapenemase-producing K. pneumonaie isolates from Germany. Data are given in % of isolates showing possession of the corresponding gene cluster. The graph shows four most frequent virulence genes identified in more than one single isolate. Figure S3. Detailed view of the ML tree concerning ST258/ST512 – carbapenemase-producing K. pneumoniae isolates from Germany, 2008-2014. The image shows a subtree of Fig. 3 containing 52 isolates of ST258 (light violett) and ST512 (grey). Colour codes of the inner ring designate the corresponding carbapenemase type, the outer designates the wzi allele (see legend). Figure S4. ML tree of NGS-based analysis of German K. pneumoniae isolates and isolates from an international collection - detailed view of the cluster ST258/ST512 isolates. The image shows a subtree of Fig. 4 containing 66 isolates of ST258 and ST512. Colour codes of the inner ring correspond to the origin of strains, the middle ring to the carbapenemase KPC-2 or KPC-3, and the outer ring demonstrates the wzi allele type. (PPTX 1367 kb

Additional file 3: of Genome-based analysis of Carbapenemase-producing Klebsiella pneumoniae isolates from German hospital patients, 2008-2014

Table S2. Names and sequences of used primers for MLST and PCR amplification of virulence genes and β-lactamase genes. Table S3. Identified virulence genes in K. pneumoniae isolate no. 316/15 (ST23, OXA-48). Table S4. Identified wzi alleles from NGS data of 107 carbapenemase-producing K. pneumoniae from Germany. (DOCX 35 kb

Table2.PDF

<p>Extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant K. pneumoniae in a German neurorehabilitation center from April 2015 to April 2016 to dissect the benefit of whole genome sequencing (WGS) for outbreak analyses. In total, 53 isolates were obtained from 52 patients and examined using WGS. Two independent analysis strategies (reference-based and -free) revealed the same distinct clusters of two CTX-M-15 producing K. pneumoniae clones (ST15, n = 31; ST405, n = 7) and one CTX-M-15 producing Klebsiella quasipneumoniae strain (ST414, n = 8). Additionally, we determined sequence variations associated with antimicrobial resistance phenotypes in single isolates expressing carbapenem and colistin resistance, respectively. For rapid detection of the major K. pneumoniae outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and K. pneumoniae genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (refRank; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (SNPfilter; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.</p

Image2.PDF

<p>Extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant K. pneumoniae in a German neurorehabilitation center from April 2015 to April 2016 to dissect the benefit of whole genome sequencing (WGS) for outbreak analyses. In total, 53 isolates were obtained from 52 patients and examined using WGS. Two independent analysis strategies (reference-based and -free) revealed the same distinct clusters of two CTX-M-15 producing K. pneumoniae clones (ST15, n = 31; ST405, n = 7) and one CTX-M-15 producing Klebsiella quasipneumoniae strain (ST414, n = 8). Additionally, we determined sequence variations associated with antimicrobial resistance phenotypes in single isolates expressing carbapenem and colistin resistance, respectively. For rapid detection of the major K. pneumoniae outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and K. pneumoniae genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (refRank; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (SNPfilter; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.</p

Table1.PDF

<p>Extended-spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae pose an important threat of infection with increased morbidity and mortality, especially for immunocompromised patients. Here, we use the rise of multidrug-resistant K. pneumoniae in a German neurorehabilitation center from April 2015 to April 2016 to dissect the benefit of whole genome sequencing (WGS) for outbreak analyses. In total, 53 isolates were obtained from 52 patients and examined using WGS. Two independent analysis strategies (reference-based and -free) revealed the same distinct clusters of two CTX-M-15 producing K. pneumoniae clones (ST15, n = 31; ST405, n = 7) and one CTX-M-15 producing Klebsiella quasipneumoniae strain (ST414, n = 8). Additionally, we determined sequence variations associated with antimicrobial resistance phenotypes in single isolates expressing carbapenem and colistin resistance, respectively. For rapid detection of the major K. pneumoniae outbreak clone (ST15), a selective triplex PCR was deduced from WGS data of the major outbreak strain and K. pneumoniae genome data deposited in central databases. Moreover, we introduce two novel open-source applications supporting reference genome selection (refRank; https://gitlab.com/s.fuchs/refRank) and alignment-based SNP-filtering (SNPfilter; https://gitlab.com/s.fuchs/snpfilter) in NGS analyses.</p