Pneumonia-Specific Escherichia coli with Distinct Phylogenetic and Virulence Profiles, France, 2012-2014.
Until the early 2000s, Enterobacteriaceae were not considered as major pathogens responsible for ventilator-assisted pneumonia (2); as such, pathophysiological studies focused mainly on Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii. However, recent data have consistently shown that Enterobacteriaceae are now frequent etiologic agents of ventilator-assisted pneumonia, more frequent than P. aeruginosa and S. aureus (4-6). According to the World Health Organization, Enterobacteriaceae, including Escherichia coli, are among the critical priority antibiotic-resistant bacteria (7). Therefore, to optimize patient management, in-depth epidemiologic knowledge of the phenotypic and genotypic characteristics of these bacteria is warranted. Although most E. coli responsible for symptomatic extraintestinal infections (8), including the urinary tract (9), bloodstream (10,11), cerebral spinal fluid (12,13), and peritoneum (14), have been extensively studied, less is known about E. coli strains responsible for pneumonia (15), especially in the context of highly virulent and resistant clones such as sequence type (ST) 69 and ST131 (16).
Our previous monocentric prospective study in France found a predominance of B2 phylogenetic group (66%) among E. coli pneumonia isolates, one third of which belonging to ST complex (STc) 127 (15). To obtain further insights in the physiopathology of ventilator-assisted pneumonia, we conducted a multicentric prospective epidemiologic study of genomic and phylogenetic characteristics of E. coli strains responsible for pneumonia across France.
Materials and Methods
Patients and E. coli Isolates
We conducted this prospective study in 14 ICUs throughout France, in collaboration with their hospital laboratories. We selected these ICUs to guarantee appropriate geographic coverage of metropolitan France. The same geographic selection was also applied to the ICUs in the Paris area. The ethics committee of the French Intensive Care Society approved the study (SRLF-CE 12-388), which was registered at ClinicalTrials.gov (NCT03303937). Formal consent was not required because of the observational, noninterventional study design (no change in practices, and all procedures already routinely performed). Patients or family/relatives were informed of the nature of the study and its purpose and objectives and had the option of declining participation.
During a 38-month period (2012-2014), any E. coli isolate responsible for pneumonia in a mechanically ventilated patient was collected, regardless of the method of sampling (quantitative cultures of tracheal suctioning, bronchoalveolar lavage, or protected telescoping catheter). Demographic and clinical data for the patient were recorded. We defined ventilator-assisted pneumonia as pneumonia occurring [greater than or equal to] 48 hours after initiation of invasive ventilation, hospital-acquired pneumonia as pneumonia occurring [greater than or equal to] 48 hours after hospital admission but within the first 48 hours of invasive ventilation, and community-acquired pneumonia as pneumonia that occurred either before or within the first 48 hours of hospitalization (17). Each E. coli isolate was stored at -80[degrees]C in brain-heart infusion broth containing glycerol 20% (the COLOCOLI collection).
E. coli Phylotyping, O-Typing, and Virulence Factor Gene Content Determination
We used quadruplex PCR to determine the E. coli phylogenetic group (A, B1, B2, C, D, E, F) or belonging to clade I (18). Among the strains, we determined the B1 clonal complex 87 (CC87) (Institut Pasteur MLST schema nomenclature corresponding to the ST58 and 155 in the Achtman schema ), the 10 main B2 subgroups (20), and the clonal group A (clonal group A) from the D phylogroup (21). The exhaustive correspondence between this typing approach and STc membership according to the currently used Achtman MLST schema is available in (22).
We used PCR to determine the most anticipated serotypes in isolates from patients with extraintestinal infections (Table 1) (23). Multiplex PCR was used to detect genes encoding for 11 frequently encountered extraintestinal virulence factors (sfa/foc, papC, papGII, papGIII, fyuA, iroN, aer, traT, neuC, hlyC, and cnf1), which belong to the main classes of virulence factors (adhesins, toxins, iron acquisition systems, and protectins) (11). For each isolate, we calculated the virulence score, which was defined by the number of virulence factors present among the 11 tested.
Antimicrobial Resistance Phenotypes
We determined the antimicrobial susceptibility of each isolate by using the disk-diffusion method according to the French Society of Microbiology (https://www.sfm-microbiologie.org) (Table 2). Resistance score was defined as the sum of inactive in vitro antimicrobial agents for each isolate. A score of 1 indicates resistant; 0.5, intermediary; and 0, sensitive. A higher score indicates a more resistant isolate. Detection of gene sequences coding for the TEM, SHV, and CTX-M enzymes was performed by PCR with genomic DNA. The oligonucleotide primer sets specific for the [beta]-lactamase genes used in the PCR assays have been published (24,25).
Characteristics of Other E. coli Strain Collections
To learn more about E. coli pneumonia strains, we compared the isolates from the COLOCOLI collection with those of 2 published collections, originating from the Paris area in France. In 2010, a total of 280 E. coli strains were isolated from fecal samples from community adults and can be considered as commensal strains (COLIVILLE collection) (26). In 2005, a total of 373 E. coli strains were isolated from the blood of 373 in-patients in 14 hospitals during the course of bacteremia (COLIBAFI collection) (27). Of note, 20.6% of isolates from patients with bacteremia were nosocomial and 57% were of urinary origin. Among patients with bacteremia, the portal of entry was pulmonary for <2% (most patients were not in an ICU).
For these strains, we determined the phylogroup/subgroup membership, the presence of the 11 virulence factors cited above, and the susceptibility to 6 antimicrobial drugs (amoxicillin, amoxicillin/clavulanic acid, cefotaxime, amikacin, ofloxacin, and cefoxitin). For all strains, we calculated a virulence score.
For our analyses we used GraphPad Prism7 software (https://www.graphpad.com). For quantitative variables, results are presented as the median and range, and for categorical variables, as frequency and proportion. Variables were compared according to whether they were nosocomial or community isolates and whether they were of phylogenetic group B2 or not B2. As we compared the virulence factor gene content of the 3 collections, we also compared the proportion of phylogenetic groups and subgroups and of resistant strains. We used the Student t test to compare continuous variables and the Fisher exact test to compare categorical variables. Because of the observational design of the study and its exploratory aim, we did not adjust for multiple testing (28). We considered p<0.05 to be significant.
During the study period, we collected 260 E. coli isolates from 243 patients with a median age of 64 years (interquartile range 52-73 years) (Table 3). Of these isolates, 117 were responsible for ventilator-assisted pneumonia, 61 for hospital-acquired pneumonia, and 82 for community acquired pneumonia. The main reasons for ICU admission were acute respiratory failure (n = 61, 25.1%), coma (n = 48, 19.8%), and septic shock (n = 44, 18.1%). A total of 98 (40.3%) patients had received antimicrobial drugs in the previous 3 months.
E. coli Characteristics
E. coli alone was isolated from 76.5% of the respiratory samples, whereas 23.5% of the samples were polymicrobial. The monomicrobial and polymicrobial samples did not differ in terms of phylogroup, virulence factor content, or antimicrobial resistance. We compiled classifications of the different phylogenetic groups/subgroups (Table 4) and details about their community or nosocomial status (Appendix Table 1, https://wwwnc.cdc.gov/EID/ article/25/4/18-0944-App1.pdf). The main phylogenetic groups were B2 (n = 155, 59.6%), B1 (n = 26, 10%), and D (n = 25, 9.6%). The most commonly identified lineages were STc73 (subgroup II, n = 40, 25.8% of B2 isolates), STc131 (subgroup I, n = 24, including 18 ST131), STc69 (clonal group A , n = 20), STc141 (subgroup IV, n = 20), and STc127 (subgroup III, n = 16). STc95 (subgroup IX) encompassed 14 strains (9% of B2 isolates). Community and nosocomial isolates did not differ in terms of phylogenetic group, except for C phylogroup isolates, which had a community predisposition (14.6% community vs. 4.5% nosocomial; p = 0.01).
We identified the O-type of 163 strains. We identified B2 phylogroup strains at the clonal level as having a combination of subgroup and O-type, as previously described (27) (Table 1). Among B2 strains, clones II-O6a were predominant, followed by IV-O2b, I-O25b (which belongs to ST131), III-O6a (which belongs to the highly virulent archetypal strain 536 ), and VI-O4.
Resistance and virulence traits of the 260 strains are detailed in Table 2 and Appendix Table 2. B2 phylogroup strains carried more virulence factor genes (virulence score 7 [5-7]) than non-B2 phylogroup strains (3 [2-4]; p<0.0001). However, traT genes were significantly more present in non-B2 (78.1%) than B2 phylogroup (45.2%) isolates (p<0.0001), as were aer genes (non-B2 63.8% and B2 41.9%; p = 0.0006). In nearly three quarters of strains, mainly B2 strains, we found iroN (70%) andfyuA (78.1%). Community and nosocomial isolates did not differ in terms of virulence and resistance scores. A total of 22 (8.5%) isolates were producers of extended-spectrum p-lactamases, including 13 CTX-M-1 group, 6 CTX-M-9 group, and 3 TEM. One isolate produced an OXA-48 carbapenemase, 28 (10.8%) isolates were resistant to cefotaxime, and 48 (18.5%) strains were resistant to piperacillin-tazobactam. B2 phylogroup strains were more sensitive to antimicrobials (resistance score 1.5 [0-4]) than were non-B2 phylogroup strains (resistance score 4.5 [2.5-7]; p<0.0001).
General Comparisons of the COLOCOLI, COLIBAFI, and COLIVILLE Collections
When comparing the pneumonia E. coli isolates with those from the 2 other collections, we found strong differences (Table 5; Figure). The B2 phylogroup was overrepresented in pneumonia strains (59.6 %) compared with commensal strains (32.1%; p<0.0001) but not with bacteremia strains (52%; p = 0.06). Among B2 phylogroup pneumonia strains, subgroup III (STc127) was significantly overrepresented (10.3%) compared with the 2 other collections (bacteremia 4.1%, p = 0.03; commensal 2.2%, p = 0.02). The proportion of subgroup IV (STc141) isolates was significantly higher among pneumonia strains (12.9%) than bacteremia strains (2.6%; p = 0.0002), whereas subgroup IX (STc95) isolates (9%) were underrepresented compared with the bacteremia B2 phylogroup strains (29.4%; p<0.0001). Within the D phylogenetic group, the proportions of clonal group A pneumonia isolates (80%) were greater than those of commensal isolates (40%; p = 0.001).
Virulence scores of pneumonia isolates (5 [3-7]) were significantly higher than those of commensal isolates (3 [1-5]; p<0.0001) but not different from those of the bacteremia isolates (4 [2-7]; p = 0.3). However, some adhesins (sfa/foc, papGIII), some toxins (hlyC, cnf1), and iroN were significantly overrepresented in pneumonia E. coli strains compared with strains in the 2 other collections.
Pneumonia E. coli isolates were more resistant than commensal isolates to all tested antimicrobial drugs except amikacin. Pneumonia isolates and bacteremia isolates did not differ in terms of antimicrobial drug susceptibility, except for resistance to cefotaxime (pneumonia isolate resistance 10.8%) and cefoxitin (pneumonia isolate resistance 11.5%) compared with bacteremia isolates (5.1%; p = 0.009 for cefotaxime, p = 0.004 for cefoxitin). Of note, when the 260 pneumonia isolates were compared with the 220 bacteremia strains of urinary tract origin, they were still distinguishable in terms of phylogroups/subgroups and virulence factor content (Appendix).
This prospective nationwide study provides data on E. coli pneumonia isolates in critically ill patients. With regard to the characteristics of E. coli pneumonia isolates, we found the following: 1) a preponderance of phylogenetic group B2 (59.6%); 2) a predominant STc73 (subgroup II) lineage and threatening emergence of ST131 (within subgroup I), STc69 (clonal group A), and STc127 (subgroup III), along with STc141 (subgroup IV); 3) a much lower proportion of STc95 (subgroup IX) in B2 pneumonia than in bacteremia isolates; 4) a specific virulence factor gene content in pneumonia versus bacteremia strains. Taken together, these epidemiologic, phylogenetic, genotypic, and experimental data argue for inclusion of E. coli pneumonia isolates in the extraintestinal pathogenic E. coli (ExPEC) pathovar but with distinguishable lung-specific traits.
In 2010, Croxen and Finlay reviewed the molecular mechanisms of E. coli pathogenicity (31). Among ExPEC, numerous pathovars were listed, including uropathogenic and neonatal meningitis pathogenic E. coli, but none concerned the lungs. Pneumonia was not even cited as a possible disease caused by E. coli.
The situation is now clearly different. In 2012, Hamet et al. reported that Enterobacteriaceae accounted for a quarter of the 323 episodes of ventilator-assisted pneumonia occurring in their ICU (6). Our group showed that over a 5-year analysis of ventilator-assisted pneumonia episodes, finding Enterobacteriaceae as the responsible pathogen increased significantly (5). In an international multicenter study, Kollef et al. also confirmed that Enterobacteriaceae were the leading pathogens of ventilator-assisted pneumonia in the ICU (4). Among them, E. coli is a major threat, recently highlighted by the World Health Organization, because of its ever-increasing resistance to antimicrobial drugs (7).
ExPEC are characterized by pathogenic virulence factor genes coding for various combinations of adhesins, toxins, iron-acquisition systems, capsule production, and toxins that enable them to cause disease once outside the host gut reservoir (32). ExPEC virulence factors are encoded on the bacterial chromosome, where they are usually located within pathogenicity-associated islands (PAIs) or plasmids. Most ExPEC isolates belong to the B2 phylogroup and, to a lesser extent, the D phylogroup. More in-depth analysis of ExPEC strains has enabled characterization of particular STs of ExPEC isolates including ST131, ST73, and ST127 (33). Our most striking finding was the specificity of pneumonia E. coli strains, compared with bacteremia ones, within the ExPEC family (Table 5). First, although B2 phylogroup strains are preponderant in ExPEC (15,34), we found a trend toward an even greater proportion of B2 isolates among pneumonia isolates (59.6%) than among bacteremia isolates, whatever the origin (52%; p = 0.06) (Table 3; Figure). This finding is in the range of what is observed in urosepsis isolates (62%) (11) but a little less than in neonatal meningitis strains (68%) (13). Then, among B2 phylogroup isolates (Table 4), if the predominance of subgroup II (STc73) was expected, we found high proportions of specific phylogenetic group B2 clones among other lineages, which could be worrisome (Table 1). The ST131 O25b:H4 clone represented 9.7% of the B2 phylogroup isolates, 53.3% of them producing an extended-spectrum P-lactamase. Subgroup IV (STc141) was significantly more present among pneumonia strains than among bacteremia strains. Challenging the hypothesis of a commensal character, with a low level of human invasiveness (27), this finding indicates that subgroup IV isolates may have a high affinity for the respiratory tract. Last, we must highlight the greater proportion of subgroup III among pneumonia isolates (namely STc127) compared with bacteremia and commensal isolates, in agreement with previous findings from our group (15). Contrary to isolates from the other 2 collections, pneumonia isolates were composed of fewer IX subgroup strains (STc95). Whereas subgroup IX is usually well represented among commensal and other pathogenic E. coli strains (26,27,35), these data suggest that the respiratory tract is less suitable than other tissues for subgroup IX implantation, a finding in agreement with our previous report in which subgroup IX was not represented (15).
Among D phylogroup strains, the higher prevalence of clonal group A (STc69) in pneumonia isolates than in bacteremia and commensal isolates was unexpected. Among 571 D phylogroup E. coli responsible for extraintestinal infection, Johnson et al. reported only 144 clonal group A E. coli (25.2%) (29). The multidrug-resistant nature of these pathogens is of increasing concern (29,36).
Regarding virulence factor gene content, the literature suggests that PAIs involved in ExPEC causing pneumonia differ from those involved in urinary tract and blood stream infections (37,38). Using PAI deletion mutants in a rat model of pneumonia, Phillips-Houlbracq et al. related pneumonia pathogenicity to the presence of PAIs I and III (37). In our study (Figure 1), pneumonia isolates differed from bacteremia isolates because they significantly more often carried sfa/foc, iroN (both belonging to PAI III), papGIII and cnf1 (belonging to PAI II), and hlyC (belonging to PAI I and II). The role of a-hemolysin in experimentally induced pneumonia in rats has been reported (15,39). Of note, the virulence factor content of the pneumonia strains still differs when we consider only the urosepsis isolates (Appendix). Although additional studies are required to confirm, these findings do suggest a coherent molecular trait for the isolates' lung specificity.
Consistent with data in the literature (15), we found that pneumonia B2 isolates were less resistant to antimicrobial drugs (B2 resistance score 1.5 [0-4] vs. non-B2 resistance score 4.5 [2.5-7]; p<0.0001) but carried more virulence factor genes (B2 virulence score 7 [5-7] vs. non-B2 virulence score 3 [2-4]; p<0.0001) (Table 2). However, contrary to an old belief, this trade-off does not mean that antimicrobial drug resistance decreases with increasing virulence (40). Indeed, virulence and antimicrobial-drug resistance were both higher in pneumonia isolates than in commensal isolates and the following were more highly represented: sfa/foc, papC, papGIII, fyuA, iroN, hlyC, and cnf1.
Our study should be interpreted within the context of its limitations. First, although our collection of E. coli pneumonia isolates is large, results regarding some subgroup analyses will require confirmation because of their sample size. Our choice of PCR rather than whole-genome characterization was governed by our wish to compare the 2 other collections with the same type of data. Despite these limitations, our analysis based on 14 centers representing France on a population and geographic level, its prospective design, and the comparison of our large number of isolates with those from recently published collections enable us to draw valid conclusions. We did not assess the functionality and expression of the encoded virulence factors in these isolates. Our team has consistently demonstrated these features in several murine models of infection (including pneumonia) and observed a strong correlation between the presence of these genes and death (15,34,37).
Our data raise the question of why certain clonal lineages were overrepresented in patients with respiratory tract infection. Patients acquire E. coli infection from their own digestive tract (41). This event implies an upward retrograde motion of the bacterial cells to reach the oropharynx and the lung parenchyma and suggests particular metabolic-adaptation and response-to-stress characteristics (e.g., to overcome the acidity of the stomach). We have previously shown that some E. coli strains are capable of high growth capacities in relation to metabolic pathways (42) while others are highly resistant to stress (43). For both studies, however, no link to specific clones could be established. The specific organ tropism therefore more probably results from a combination of genetic background and virulence factors.
In summary, we identified emerging pneumonia-causing pathogenic E. coli whose main characteristics define them as ExPEC. Their specificities include a very strong proportion of B2 phylogroup isolates; a high proportion of subgroups II (STc73), I (STc131), IV (STc141), and III (STc127); and consequent proportions of clonal group A (STc69) isolates within the D phylogroup. Virulence factor gene content of pneumonia isolates also appeared to be singular compared with that of bacteremia isolates, among them urosepsis isolates. These epidemiologic data underline the specificity of pneumonia E. coli populations and may help with the design of more targeted therapies.
We are indebted to the physicians (microbiologists and intensivists) who took part in the COLOCOLI Study. We are also grateful to the patients and their families, and we gratefully acknowledge Dominique Poignard and Marie-Claire Hipeaux for technical assistance and all the nurses and assistant nurses of the ICU for their help in obtaining the microbiological samples.
Collaborators for the COLOCOLI group: Catherine Branger, Luce Landraud, Alexandre Bleibtreu, Franjoise Jaureguy (Infection, Antimicrobiens, Modelisation, Evolution, Paris, France); Pierre Asfar (Centre Hospitalier Universitaire, Angers, France); Didier Dreyfuss, Guilene Barnaud, Fatma Magdoud (Hopital Louis Mourier, AP-HP, Colombes, France); Michel Wolff, Jean-Franjois Timsit (Hopital Bichat, AP-HP, Paris, France); Stephanie Martin (Centre Hospitalier Departemental Vendee, La Roche-sur-Yon, France); Pierre-Emmanuel Charles (Centre Hospitalier Universitaire Bocage Central, Dijon, France); Alexandre Boyer (Centre Hospitalier Universitaire Pellegrin, Bordeaux, France); Emmanuelle Jaillette, Saad Nseir (Centre Hospitalier Regional Universitaire, Lille, France); Raymond Ruimy, Pierre-Eric Danin, Jean Dellamonica (Centre Hospitalier Universitaire, Nice, France); Julie Cremniter, Jean-Pierre Frat (Centre Hospitalier Universitaire, Poitiers, France); Christophe Clec'h (Hopital Avicenne, AP-HP, Bobigny, France), Dominique Decre, Eric Maury (Hopital Saint-Antoine, AP-HP, Paris, France).
This work was supported by a grant from the Fonds de Dotation Recherche en Sante Respiratoire/Fondation du Souffle to (B.L.C.). This work was partly supported by a grant from the Fondation pour la Recherche Medicale (to E.D., Equipe FRM 2016, grant no. DEQ20161136698).
Dr. La Combe is an intensive care physician who works at the Infection, Antimicrobials, Modelling, Evolution laboratory of the French Institute for Medical Research in Paris. Her work focuses on the virulence and antimicrobial resistance of respiratory E. coli isolates and the pharmacodynamics and genetics of chlorhexidine resistance of oropharyngeal isolates in patients receiving mechanical ventilation.
(1.) Melsen WG, Rovers MM, Groenwold RHH, Bergmans DCJJ, Camus C, Bauer TT, et al. Attributable mortality of ventilator-associated pneumonia: a meta-analysis of individual patient data from randomised prevention studies. Lancet Infect Dis. 2013;13:665-71. http://dx.doi.org/10.1016/S1473-3099 (13)70081-1
(2.) Chastre J, Fagon J-Y. Ventilator-associated pneumonia. Am J Respir Crit Care Med. 2002;165:867-903. http://dx.doi.org/ 10.1164/ajrccm.165.7.2105078
(3.) Russo TA, Johnson JR. Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes Infect. 2003;5:449-56. http://dx.doi.org/10.1016/S1286-4579(03)00049-2
(4.) Kollef MH, Ricard J-D, Roux D, Francois B, Ischaki E, Rozgonyi Z, et al. A randomized trial of the amikacin fosfomycin inhalation system for the adjunctive therapy of gram-negative ventilator-associated pneumonia: IASIS Trial. Chest. 2017;151:1239-46. http://dx.doi.org/10.1016/j.chest.2016.11.026
(5.) Fihman V, Messika J, Hajage D, Tournier V, Gaudry S, Magdoud F, et al. Five-year trends for ventilator-associated pneumonia: correlation between microbiological findings and antimicrobial drug consumption. Int J Antimicrob Agents. 2015;46:518-25. http://dx.doi.org/10.1016/j.ijantimicag. 2015.07.010
(6.) Hamet M, Pavon A, Dalle F, Pechinot A, Prin S, Quenot J-P, et al. Candida spp. airway colonization could promote antibiotic-resistant bacteria selection in patients with suspected ventilator associated pneumonia. Intensive Care Med. 2012;38:1272-9. http://dx.doi.org/10.1007/s00134-012-2584-2
(7.) World Health Organization. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics [cited 2017 Sep 29]. http://www.who.int/ medicines/publications/global-priority-list-antibiotic-resistantbacteria/en/
(8.) Johnson JR, Russo TA. Extraintestinal pathogenic Escherichia coli: "the other bad E coli." J Lab Clin Med. 2002;139:155-62. http://dx.doi.org/10.1067/mlc.2002.121550
(9.) Johnson JR, Kuskowski MA, Gajewski A, Soto S, Horcajada JP, Jimenez de Anta MT, et al. Extended virulence genotypes and phylogenetic background of Escherichia coli isolates from patients with cystitis, pyelonephritis, or prostatitis. J Infect Dis. 2005;191:46-50. http://dx.doi.org/10.1086/426450
(10.) Johnson JR, Kuskowski MA, O'Bryan TT, Maslow JN. Epidemiological correlates of virulence genotype and phylogenetic background among Escherichia coli blood isolates from adults with diverse-source bacteremia. J Infect Dis. 2002;185:1439-47. http://dx.doi.org/10.1086/340506
(11.) Lefort A, Panhard X, Clermont O, Woerther P-L, Branger C, Mentre F, et al.; COLIBAFI Group. Host factors and portal of entry outweigh bacterial determinants to predict the severity of Escherichia coli bacteremia. J Clin Microbiol. 2011;49:777-83. http://dx.doi.org/10.1128/JCM.01902-10
(12.) Bidet P, Mahjoub-Messai F, Blanco J, Blanco J, Dehem M, Aujard Y, et al. Combined multilocus sequence typing and O serogrouping distinguishes Escherichia coli subtypes associated with infant urosepsis and/or meningitis. J Infect Dis. 2007;196:297-303. http://dx.doi.org/10.1086/518897
(13.) Bingen E, Picard B, Brahimi N, Mathy S, Desjardins P, Elion J, et al. Phylogenetic analysis of Escherichia coli strains causing neonatal meningitis suggests horizontal gene transfer from a predominant pool of highly virulent B2 group strains. J Infect Dis. 1998;177:642-50. http://dx.doi.org/10.1086/514217
(14.) Bert F, Johnson JR, Ouattara B, Leflon-Guibout V, Johnston B, Marcon E, et al. Genetic diversity and virulence profiles of Escherichia coli isolates causing spontaneous bacterial peritonitis and bacteremia in patients with cirrhosis. J Clin Microbiol. 2010;48:2709-14. http://dx.doi.org/10.1128/ JCM.00516-10
(15.) Messika J, Magdoud F, Clermont O, Margetis D, Gaudry S, Roux D, et al. Pathophysiology of Escherichia coli ventilator-associated pneumonia: implication of highly virulent extra-intestinal pathogenic strains. Intensive Care Med. 2012;38:2007-16. http://dx.doi.org/10.1007/s00134-012-2699-5
(16.) Blanco J, Mora A, Mamani R, Lopez C, Blanco M, Dahbi G, et al. National survey of Escherichia coli causing extra-intestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain. J Antimicrob Chemother. 2011;66:2011-21. http://dx.doi.org/10.1093/jac/dkr235
(17.) American Thoracic Society, Infectious Diseases Society of America. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med. 2005;171:388-416. http://dx.doi.org/10.1164/rccm.200405-644ST
(18.) Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013;5:58-65. http://dx.doi.org/ 10.1111/1758-2229.12019
(19.) Skurnik D, Clermont O, Guillard T, Launay A, Danilchanka O, Pons S, et al. Emergence of antimicrobial-resistant Escherichia coli of animal origin spreading in humans. Mol Biol Evol. 2016;33:898-914. http://dx.doi.org/10.1093/molbev/msv280
(20.) Clermont O, Christenson JK, Daubie A-S, Gordon DM, Denamur E. Development of an allele-specific PCR for Escherichia coli B2 sub-typing, a rapid and easy to perform substitute of multilocus sequence typing. J Microbiol Methods. 2014;101:24-7. http://dx.doi.org/10.1016/j.mimet.2014.03.008
(21.) Johnson JR, Owens K, Manges AR, Riley LW. Rapid and specific detection of Escherichia coli clonal group A by gene-specific PCR. J Clin Microbiol. 2004;42:2618-22. http://dx.doi.org/10.1128/ JCM.42.6.2618-2622.2004
(22.) Clermont O, Gordon D, Denamur E. Guide to the various phylogenetic classification schemes for Escherichia coli and the correspondence among schemes. Microbiology. 2015;161:980-8. http://dx.doi.org/10.1099/mic.0.000063
(23.) Clermont O, Johnson JR, Menard M, Denamur E. Determination of Escherichia coli O types by allele-specific polymerase chain reaction: application to the O types involved in human septicemia. Diagn Microbiol Infect Dis. 2007;57:129-36. http://dx.doi.org/10.1016/j.diagmicrobio.2006.08.007
(24.) Eckert C, Gautier V, Saladin-Allard M, Hidri N, Verdet C, Ould-Hocine Z, et al. Dissemination of CTX-M-type betalactamases among clinical isolates of Enterobacteriaceae in Paris, France. Antimicrob Agents Chemother. 2004;48:1249-55. http://dx.doi.org/10.1128/AAC.48.4.1249-1255.2004
(25.) Rasheed JK, Jay C, Metchock B, Berkowitz F, Weigel L, Crellin J, et al. Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia. Antimicrob Agents Chemother. 1997; 41:647-53. http://dx.doi.org/10.1128/AAC.4L3.647
(26.) Massot M, Daubie A-S, Clermont O, Jaureguy F, Couffignal C, Dahbi G, et al.; The COLIVILLE Group. Phylogenetic, virulence and antibiotic resistance characteristics of commensal strain populations of Escherichia coli from community subjects in the Paris area in 2010 and evolution over 30 years. Microbiology. 2016;162:642-50. http://dx.doi.org/10.1099/mic.0.000242
(27.) Clermont O, Couffignal C, Blanco J, Mentre F, Picard B, Denamur E; COLIVILLE and COLIBAFI groups. Two levels of specialization in bacteraemic Escherichia coli strains revealed by their comparison with commensal strains. Epidemiol Infect. 2017;145:872-82. http://dx.doi.org/10.1017/S0950268816003010
(28.) Bender R, Lange S. Adjusting for multiple testing--when and how? J Clin Epidemiol. 2001;54:343-9. http://dx.doi.org/10.1016/ S0895-4356(00)00314-0
(29.) Johnson JR, Menard ME, Lauderdale T-L, Kosmidis C, Gordon D, Collignon P, et al.; Trans-Global Initiative for Antimicrobial Resistance Analysis Investigators. Global distribution and epidemiologic associations of Escherichia coli clonal group A, 1998-2007. Emerg Infect Dis. 2011;17:2001-9. http://dx.doi.org/ 10.3201/eid1711.110488
(30.) Brzuszkiewicz E, Bruggemann H, Liesegang H, Emmerth M, Olschlager T, Nagy G, et al. How to become a uropathogen: comparative genomic analysis of extraintestinal pathogenic Escherichia coli strains. Proc Natl Acad Sci U S A. 2006; 103:12879-84. http://dx.doi.org/10.1073/pnas.0603038103
(31.) Croxen MA, Finlay BB. Molecular mechanisms of Escherichia coli pathogenicity. Nat Rev Microbiol. 2010;8:26-38. http://dx.doi.org/10.1038/nrmicro2265
(32.) Russo TA, Johnson JR. Proposal for a new inclusive designation for extraintestinal pathogenic isolates of Escherichia coli: ExPEC. J Infect Dis. 2000;181:1753-4. http://dx.doi.org/10.1086/ 315418
(33.) Dale AP, Woodford N. Extra-intestinal pathogenic Escherichia coli (ExPEC): disease, carriage and clones. J Infect. 2015;71:615-26. http://dx.doi.org/10.1016/jJinf.2015.09.009
(34.) Picard B, Garcia JS, Gouriou S, Duriez P, Brahimi N, Bingen E, et al. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun. 1999;67:546-53.
(35.) Ciesielczuk H, Jenkins C, Chattaway M, Doumith M, Hope R, Woodford N, et al. Trends in ExPEC serogroups in the UK and their significance. Eur J Clin Microbiol Infect Dis. 2016;35:1661-6. http://dx.doi.org/10.1007/ s10096-016-2707-8
(36.) Kallonen T, Brodrick HJ, Harris SR, Corander J, Brown NM, Martin V, et al. Systematic longitudinal survey of invasive Escherichia coli in England demonstrates a stable population structure only transiently disturbed by the emergence of ST131. Genome Res. 2017;27:1437-49. http://dx.doi.org/10.1101/ gr.216606.116
(37.) Phillips-Houlbracq M, Ricard J-D, Foucrier A, Yoder-Himes D, Gaudry S, Bex J, et al. Pathophysiology of Escherichia coli pneumonia: respective contribution of pathogenicity islands to virulence. Int J Med Microbiol. 2018;308:290-6. http://dx.doi.org/ 10.1016/j.ijmm.2018.01.003
(38.) Tourret J, Diard M, Garry L, Matic I, Denamur E. Effects of single and multiple pathogenicity island deletions on uropathogenic Escherichia coli strain 536 intrinsic extra-intestinal virulence. Int J Med Microbiol. 2010;300:435-9. http://dx.doi.org/10.1016/ j.ijmm.2010.04.013
(39.) Russo TA, Davidson BA, Genagon SA, Warholic NM, Macdonald U, Pawlicki PD, et al. E. coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model. Am J Physiol Lung Cell Mol Physiol. 2005;289:L207-16. http://dx.doi.org/10.1152/ajplung.00482.2004
(40.) Roux D, Danilchanka O, Guillard T, Cattoir V, Aschard H, Fu Y, et al. Fitness cost of antibiotic susceptibility during bacterial infection. Sci Transl Med. 2015;7:297ra114. http://dx.doi.org/ 10.1126/scitranslmed.aab1621
(41.) Garrouste-Orgeas M, Chevret S, Arlet G, Marie O, Rouveau M, Popoff N, et al. Oropharyngeal or gastric colonization and nosocomial pneumonia in adult intensive care unit patients. A prospective study based on genomic DNA analysis. Am J Respir Crit Care Med. 1997;156:1647-55. http://dx.doi.org/10.1164/ ajrccm.156.5.96-04076
(42.) Sabarly V, Bouvet O, Glodt J, Clermont O, Skurnik D, Diancourt L, et al. The decoupling between genetic structure and metabolic phenotypes in Escherichia coli leads to continuous phenotypic diversity. J Evol Biol. 2011;24:1559-71. http://dx.doi.org/10.1111/j.1420-9101.2011.02287.x
(43.) Bleibtreu A, Gros P-A, Laouenan C, Clermont O, Le Nagard H, Picard B, et al. Fitness, stress resistance, and extraintestinal virulence in Escherichia coli. Infect Immun. 2013;81:2733-42. http://dx.doi.org/10.1128/IAI.01329-12
Address for correspondence: Jean-Damien Ricard, Service de Reanimation Medicale, Hopital Louis Mourier, 92700 Colombes, France; email: email@example.com
Author affiliations: Infection, Antimicrobiens, Modelisation, Evolution, Paris, France (B. La Combe, O. Clermont, J. Messika, T. Billard-Pomares, R. Fernandes, L. Armand-Lefevre, E. Denamur, J.-D. Ricard); Universite Paris Diderot, Paris (B. La Combe, O. Clermont, J. Messika, T. Billard-Pomares, R. Fernandes, L. Armand-Lefevre, E. Denamur, J.-D. Ricard); Hopital Louis Mourier, Colombes, France (B. La Combe, J. Messika, T. Billard-Pomares, R. Fernandes, J.-D. Ricard); Centre Hospitalier Universitaire, Angers, France (M. Eveillard, A. Kouatchet, S. Lasocki); Centre Hospitalier Universitaire, Nantes, France (S. Corvec, J. Reignier); Hopital Laennec, Nantes (K. Lakhal); Hopital Bichat, AP-HP, Paris (L. Armand-Lefevre, E. Denamur); Centre Hospitalier Departemental Vendee, La Roche-sur-Yon, France (S. Bourdon); Hopital Henri Mondor, AP-HP, Creteil, France (V. Fihman, N. de Prost); Centre Hospitalier Universitaire Bocage Central, Dijon, France (J. Bador); Centre Hospitalier Universitaire Pellegrin, Bordeaux, France (J. Goret); Centre Hospitalier Regional Universitaire, Lille, France (F. Wallet)
Beatrice La Combe, Olivier Clermont, Jonathan Messika, Matthieu Eveillard, Achille Kouatchet, Sigismond Lasocki, Stephane Corvec, Karim Lakhal, Typhaine Billard-Pomares, Romain Fernandes, Laurence Armand-Lefevre, Sandra Bourdon, Jean Reignier, Vincent Fihman, Nicolas de Prost, Julien Bador, Julien Goret, Frederic Wallet, Erick Denamur, Jean-Damien Ricard, on behalf of the COLOCOLI group 
 Group members are listed at the end of this article.
Caption: Figure. Comparison of Escherichia coli isolates among pneumonia patients with extraintestinal infections, France, 2012-2014, with commensal E. coli (COLIVILLE collection) and E. coli bacteremia isolates (COLIBAFI collection). A) Proportions of E. coli phylogenetic groups and subgroups; B) B2 subgroups, D CGA, and B1 CC87; C) virulence factors; and D) antimicrobial drug resistance. Roman numerals correspond to the B2 subgroup. Correspondence with the Achtman multilocus sequence typing schema is as follows: subgroup I, STc131; subgroup II, STc73; subgroup III, STc127; subgroup IV, STc141; subgroup VI, STc12; subgroup VII, STc14; subgroup IX, STc95 (22). Proportions of subgroups are reported as fractions of the respective phylogroups. Asterisks indicate a significant difference between respiratory isolates and strains responsible for bacteremia (COLIBAFI collection) or commensal strains (COLIVILLE collection): *p<0.05; **p<0.005; ***p<0.0005. AMC, amoxicillin/clavulanic acid; AMK, amikacin; AMX, amoxicillin; CTX, cefotaxime; FOX, cefoxitin; OFX, ofloxacin; STc, sequence type complex.
Table 1. Characterization of the main Escherichia coli B2 phylogroup clones, by combination of subgroup and O-type, among patients with extraintestinal infections, France, 2012-2014 * Subqroup and O-type B2 clones, no. (%), n = 155 I-O25b 15 (62.5) I-O6a 2 (8.3) I-O16 3 (12.5) II-O22 2 (5) II-O2b 4 (10) II-O6a 26 (65) III-O6a 16 (100) IV-O2b 19 (95) VI-O4 13 (92.9) VII-O18 2 (66.6) VII-O75 1 (33.3) IX-O1 7 (50) IX-O18 3(21.4) IX-O2a 3(21.4) * Data are presented as no. (%) of each clone within the corresponding subgroup. Roman numerals correspond to the B2 subgroup. The correspondence with the Achtman multilocus sequence type schema is as follows: subgroup I, sequence type complex (STc) 131; subgroup II, STc73; subgroup III, sTc127; subgroup IV, STc141; subgroup VI, STc12; subgroup VII, STc14; subgroup IX, STc95 (22). The most anticipated serotypes in extraintestinal infections were searched by PCR: O1, O2a, O2b, O4, O6, O7, O12, O15, O16, O17, O18, O22, O25a, O25b, O45a, O75, O78. Table 2. Resistance and virulence traits of the 260 Escherichia coli isolates responsible for pneumonia in patients receiving mechanical ventilation, according to phylogenetic group, France, 2012-2014 * Phylogenetic group Trait B2, Non-B2, p value n = 155 n = 105 iroN 130 (83.9) 52 (49.5) <0.0001 sfa/foc 109 (70.3) 0 <0.0001 neuC 41 (26.4) 0 <0.0001 fyuA 152 (98.1) 51 (48.6) <0.0001 hlyC 98 (63.2) 2 (1.9) <0.0001 cnfl 91 (58.7) 1 (1) <0.0001 aer 65 (41.9) 67 (63.8) 0.0006 papC 100 (64.5) 27 (25.7) <0.0001 papGIl 27 (17.4) 4 (3.8) 0.0007 papGIII 64 (41.3) 0 <0.0001 traT 70 (45.2) 82 (78.1) <0.0001 Virulence score, median 7 (5-7) 3 (2-4) <0.0001 (IQR) ([dagger]) Antimicrobial resistance Amoxicillin 75 (48.4) 83 (79) <0.0001 Amoxicillin/clavulanic acid 66 (42.6) 68 (64.8) 0.0006 Piperacillin/tazobactam 21 (13.5) 27 (25.7) 0.02 Cefotaxime 11 (7.1) 17 (16.2) 0.02 Ceftazidime 12 (7.7) 17 (16.2) 0.04 Imipenem 0 1 (1) 0.4 Gentamicin 4 (2.6) 10 (9.5) 0.02 Amikacin 3 (1.9) 1 (1) 0.6 Ofloxacin 15 (9.7) 28 (26.7) 0.0005 Ciprofloxacin 13 (8.4) 24 (22.9) 0.002 Resistance score, median 1.5 (0-4) 4.5 (2.5-7) <0.0001 (IQR) ([double dagger]) ESBL phenotype 10 (6.4) 12 (11.4) 0.2 WT phenotype 84 (54.2) 24 (22.9) <0.0001 Phylogenetic group Trait A, B1, C, n = 22 n = 26 n = 20 iroN 10 (45.4) 13 (50) 16 (80) sfa/foc 0 0 0 neuC 0 0 0 fyuA 7(31.8) 9 (34.6) 16 (80) hlyC 0 2 (7.7) 0 cnfl 0 1 (3.8) 0 aer 14 (63.6) 15 (57.7) 14 (70) papC 7(31.8) 3 (11.5) 8 (40) papGIl 0 0 0 papGIII 0 0 0 traT 15 (68.2) 20 (76.9) 17 (85) Virulence score, median 2.5 (2-4) 3 (1-4) 4 (3-5) (IQR) ([dagger]) Antimicrobial resistance Amoxicillin 19 (86.4) 19 (73) 16 (80) Amoxicillin/clavulanic acid 18 (81.8) 13 (50) 15 (75) Piperacillin/tazobactam 6 (27.3) 6 (23.1) 7 (35) Cefotaxime 4 (18.2) 4 (15.4) 2 (10) Ceftazidime 4 (18.2) 4 (15.4) 2 (10) Imipenem 0 1 (3.8) 0 Gentamicin 3 (13.6) 1 (3.8) 2 (10) Amikacin 0 0 0 Ofloxacin 7(31.8) 6 (23.1) 6 (30) Ciprofloxacin 7(31.8) 5 (19.2) 6 (30) Resistance score, median 5 (3.5-8) 4 (1-6) 4.5 (3.5-7.5) (IQR) ([double dagger]) ESBL phenotype 4 (18.2) 1 (3.8) 1 (4) WT phenotype 4 (18.2) 7 (26.9) 6(24) Trait D, F, n = 25 n = 11 iroN 6 (24) 6 (54.5) sfa/foc 0 0 neuC 0 0 fyuA 10 (40) 8 (72.7) hlyC 0 0 cnfl 0 0 aer 15 (60) 8 (72.7) papC 8 (32) 1 (9.1) papGIl 4 (16) 0 papGIII 0 0 traT 20 (80) 9(81.8) Virulence score, median 3 (2-3) 3 (2.5-3) (IQR) ([dagger]) Antimicrobial resistance Amoxicillin 19 (76) 10 (90.9) Amoxicillin/clavulanic acid 14 (56) 8 (72.7) Piperacillin/tazobactam 3 (12) 5 (45.4) Cefotaxime 3 (12) 4 (36.4) Ceftazidime 3 (12) 4 (36.4) Imipenem 0 0 Gentamicin 1 (4) 3 (27.3) Amikacin 0 1 (9.1) Ofloxacin 2 (8) 6 (54.5) Ciprofloxacin 2 (8) 4 (36.4) Resistance score, median 4 (1.5-5.5) 7.5 (5-9) (IQR) ([double dagger]) ESBL phenotype 2 (8) 3 (27.3) WT phenotype 7(28) 1 (9.1) * Values are no. (%) except as indicated. ESBL, extended-spectrum beta-lactamase; IQR, interquartile range; WT, wild type (susceptible to all tested antimicrobials). ([dagger]) Virulence score was calculated, defined by the number of present virulence factors among the 11 tested. ([double dagger]) Resistance score was defined as the sum of inactive in vitro antimicrobial agents for each isolate. Tested antimicrobials were gentamicin, amikacin, minocycline, nalidixic acid, ofloxacin, ciprofloxacin, fosfomycin, furans, trimethoprim, amoxicillin, amoxicillin-clavulanic acid, ticarcillin, piperacillin, piperacillin-tazobactam, imipenem, cefotaxime, and ceftazidime. A score of 1 was attributed for a resistant, 0.5 for an intermediary, and 0 for a sensitive isolate; a higher score thus indicated a more resistant isolate. For each antimicrobial, resistance is defined by the sum of resistant or intermediary isolates. Table 3. Demographics and clinical characteristics of 243 pneumonia patients requiring mechanical ventilation, from whom Escherichia coli was isolated, France, 2012-2014 * Characteristic Value Age, y, median (IQR) 64 (52-73) Sex M 183 (75.3) F 60 (24.7) SAPS II at admission, median (IQR) 57 (42-69) Comorbid conditions Chronic alcohol consumption 56 (23) Diabetes mellitus 45 (18.5) Neoplastic disease 43 (17.7) Immunosuppression ([dagger]) 77 (31.7) Cirrhosis 12 (4.9) Chronic kidney disease 18 (7.4) Dialysis 5 (2.1) Chronic respiratory disease 33 (13.6) Chronic heart failure 43 (17.7) Reason for ICU admission Acute respiratory failure 61 (25.1) Coma 48 (19.8) Septic shock 44 (18.1) Cardiac arrest 28 (11.5) Cardiogenic shock 14 (5.8) Polytrauma 22 (9.1) Postoperative care 8 (3.3) Hemorrhaqic shock 5 (2.1) Exposure to antimicrobial drug therapy in 98 (40.3) previous 3 mo Amoxicillin 6 (2.5) Amoxicillin/clavulanic acid 38 (15.6) Third-qeneration cephalosporin 19 (7.8) Aminoglycosides 29 (11.9) Piperacillin/tazobactam 24 (9.9) Quinolone 10 (4.1) Carbapenem 11 (4.5) Polymicrobial sampling 57 (23.5) ICU length of stay, d (IQR) 17 (7-33) Hospital length of stay, d (IQR) 24 (10-45) Death While in ICU 90 (37) While in hospital 99 (40.7) Associated with E. coli 27 (11.1) * Values are no. (%) except as indicated. ICU, intensive care unit; IQR, interquartile range; SAPS II, Simplified Acute Physiology Score II. ([dagger]) Defined by [greater than or equal to] 1 immunosuppression factor among neoplastic disease, hematologic malignancy, HIV infection, immunosuppressive therapy, corticosteroids therapy. Table 4. Phylogenetic groups/subgroups of Escherichia coli isolated from 260 pneumonia patients requiring mechanical ventilation, France, 2012-2014 * Phylogroup No. (%) A 22 (8.5) B1 26 (10) CC87 ([dagger]) 11 (42.3) Non-CC87 15 (57.7) B2 155 (59.6) I ST131 ([dagger]) 18 (11.6) I non-ST131 6 (3.9) II 40 (25.8) III 16 (10.3) IV 20 (12.9) V 1 (0.6) VI 14 (9) VII 3 (1.9) IX 14 (9) Unassiqned 23 (14.8) C 20 (7.7) D 25 (9.6) CGA ([dagger]) 20 (80) Non-CGA 5 (20) F 11 (4.2) Clade I 1 (0.4) * Nosocomial isolates encompass ventilator-associated pneumonia and hospital-acquired pneumonia isolates. CC87, clonal complex 87 (sequence type complex [STc] 155) (19); CGA, clonal group A (STc69) (21); ST131, sequence type 131. The correspondence with the Achtman MLST schema is as follows: subgroup I, STc131; subgroup II, STc73; subgroup III, STc127; subgroup IV, STc141; subgroup V, STc144; subgroup VI,sTc12; subgroup VII, STc14; subgroup IX, STc95. No isolate belonged to subgroup VIII (STc452), X (STc372), or E phylogroup (22). ([dagger]) Proportions of subgroups are reported as fractions of the Respective phylogroups. Table 5. Proportion of phylogenetic groups, B2 subgroups, D CGA, and B1 CC87 among pneumonia, bacteremia, or commensal isolates * Phylogenetic Pneumonia Bacteremia Commensal characteristics isolates isolates isolates A - + ++ B1 + - + CC87 ([dagger]) +++ +++ + B2 ++++ ++++ +++ I + + + II ++ ++ ++ III + - - IV + - + IX - ++ + C - - - D - + + CGA * ++++ ++++ +++ E - - - F - - - * CGA, clonal group A (sequence type complex [STc] 69) (21); CC87, clonal complex 87 (STc155) (19); -, <10%; +, 10%-20%; ++, 20%-30%; +++ 30- 50%; ++++, >50% of isolates. Correspondence with the Achtman multilocus sequence typing schema is subgroup I = STc131; subgroup II = STc73; subgroup III = STc127; subgroup IV = STc141; and subgroup IX = STc95 (22). ([dagger]) Proportions of subgroups are calculated within each respective phylogroup.
|Printer friendly Cite/link Email Feedback|
|Author:||La Combe, Beatrice; Clermont, Olivier; Messika, Jonathan; Eveillard, Matthieu; Kouatchet, Achille; L|
|Publication:||Emerging Infectious Diseases|
|Article Type:||Clinical report|
|Date:||Apr 1, 2019|
|Previous Article:||Staphylococcus aureus Bacteremia in Children of Rural Areas of The Gambia, 2008-2015.|
|Next Article:||Symptoms, Sites, and Significance of Mycoplasma genitalium in Men Who Have Sex with Men.|