Contribution of Clinical Metagenomics to the Diagnosis of Bone and Joint Infections.

Bone and joint infections (BJIs) are complex infections that require precise microbiological documentation to optimize antibiotic therapy. Currently, diagnosis is based on microbiological culture, sometimes complemented by amplification and sequencing of the 16S rDNA gene. Clinical metagenomics (CMg), that is, the sequencing of the entire nucleic acids in a sample, was previously shown to identify bacteria not detected by conventional methods, but its actual contribution to the diagnosis remains to be assessed, especially with regard to 16S rDNA sequencing. In the present study, we tested the performance of CMg in 34 patients (94 samples) with suspected BJIs, as compared to culture and 16S rDNA sequencing. A total of 94 samples from 34 patients with suspicion of BJIs, recruited from two sites, were analyzed by (i) conventional culture, (ii) 16S rDNA sequencing (Sanger method), and (iii) CMg (Illumina Technology). Two negative controls were also sequenced by CMg for contamination assessment. Based on the sequencing results of negative controls, 414 out of 539 (76.7%) bacterial species detected by CMg were considered as contaminants and 125 (23.2%) as truly present. For monomicrobial infections (13 patients), the sensitivity of CMg was 83.3% as compared to culture, and 100% as compared to 16S rDNA. For polymicrobial infections (13 patients), the sensitivity of CMg was 50% compared to culture, and 100% compared to 16S rDNA. For samples negative in culture (8 patients, 21 samples), CMg detected 11 bacteria in 10 samples from 5 different patients. In 5/34 patients, CMg brought a microbiological diagnosis where conventional methods failed, and in 16/34 patients, CMg provided additional information. Finally, 99 antibiotic resistance genes were detected in 24 patients (56 samples). Provided sufficient genome coverage (87.5%), a correct inference of antibiotic susceptibility was achieved in 8/8 bacteria (100%). In conclusion, our study demonstrated that the CMg provides complementary and potentially valuable data to conventional methods of BJIs diagnosis.

Temocillin as an alternative treatment for acute bacterial cholangitis: a retrospective microbiology susceptibility-based study of 140 episodes.

With rising antibiotic resistance, alternatives to carbapenems are needed for acute cholangitis (AC). Temocillin reaches high biliary concentrations with limited impact on microbiota. We retrospectively included 140 AC episodes and assessed the efficacy of temocillin using microbiology susceptibility testing from blood cultures. Considering all bacteria collected by episode, resistance to temocillin, PIP/TAZ and 3GC occurred in 27/140 (26%), 32 (22.8%) and 31 (22%) episodes, respectively (p = 0.7). After documentation, temocillin could have spared PIP/TAZ or carbapenems in 14/26 and 4/11 episodes. Temocillin may constitute an alternative treatment after microbiological documentation by sparing carbapenems and/or PIP/TAZ, but not as an empirical therapeutic option.

Success of Escherichia coli O25b:H4 Sequence Type 131 Clade C Associated with a Decrease in Virulence.

Escherichia coli O25b:H4 sequence type 131 (ST131), which is resistant to fluoroquinolones and which is a producer of CTX-M-15, is globally one of the major extraintestinal pathogenic E. coli (ExPEC) lineages. Phylogenetic analyses showed that multidrug-resistant ST131 strains belong to clade C, which recently emerged from clade B by stepwise evolution. It has been hypothesized that features other than multidrug resistance could contribute to this dissemination since other major global ExPEC lineages (ST73 and ST95) are mostly antibiotic susceptible. To test this hypothesis, we compared early biofilm production, presence of ExPEC virulence factors (VFs), and in vivo virulence in a mouse sepsis model in 19 and 20 epidemiologically relevant strains of clades B and C, respectively. Clade B strains were significantly earlier biofilm producers (P < 0.001), carriers of more VFs (P = 4e-07), and faster killers of mice (P = 2e-10) than clade C strains. Gene inactivation experiments showed that the H30-fimB and ibeART genes were associated with in vivo virulence. Competition assays in sepsis, gut colonization, and urinary tract infection models between the most anciently diverged strain (B1 subclade), one C1 subclade strain, and a B4 subclade recombining strain harboring some clade C-specific genetic events showed that the B1 strain always outcompeted the C1 strain, whereas the B4 strain outcompeted the C1 strain, depending on the mouse niches. All these findings strongly suggest that clade C evolution includes a progressive loss of virulence involving multiple genes, possibly enhancing overall strain fitness by avoiding severe infections, even if it comes at the cost of a lower colonization ability.

High Prevalence of ST131 Subclades C2-H30Rx and C1-M27 Among Extended-Spectrum ß-Lactamase-Producing Escherichia coli Causing Human Extraintestinal Infections in Patients From Two Hospitals of Spain and France During 2015.

The present study was carried out to evaluate the prevalence of sequence type 131 (ST131) among 188 extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) collected in 2015 in Lucus Augusti University hospital (Lugo, Spain) and AP-HP Beaujon hospital (Clichy, France) with regard to other STs and to characterize, the types of ESBL produced, serotypes, virulence factor (VF)-encoding genes and the ST131 clades and subclades. ST131 was detected in 33 (39.1%) and 46 (47.9%) of the isolates in Lucus Augusti and Beaujon, respectively. The 109 remaining isolates displayed 57 other STs, the following STs being displayed by at least three isolates: ST10 (8 isolates), ST23 (3), ST38 (4), ST58 (3), ST88 (5), ST95 (4), ST167 (3), ST354 (5), ST361 (3), ST410 (6), ST648 (4), ST744 (3), and ST1615 (6). ST354, ST410, and ST1615 were significantly (P < 0.05) more frequent in Lucus Augusti (5.4%, 6.5%, and 6.5%) than in Beaujon (0% for the three STs). The new globally emerging clone ST1193 among extraintestinal clinical ESBL-EC was identified in one isolate from France and one from Spain. CTX-M-15 was the commonest ESBL detected in the two hospitals (44.6% in Lucus Augusti and 50.0% in Beaujon). CTX-M-14 was significantly (P = 0.0003) more frequent in Lucus Augusti (31.5%) than in Beaujon (10.4%), whereas CTX-M-1 (20.8 vs. 7.6%; P = 0.008) and CTX-M-27 (15.6 vs. 6.5%; P = 0.0389) were more frequent in Beaujon than in Lucus Augusti. The ST131 isolates showed a higher virulence score (mean 13.367) compared with the non-ST131 isolates (mean 7.661) (P < 0.001). Among the 79 ST131 isolates, most of them (52; 65.8%) belonged to subclade C2 (also known as subclone H30Rx) followed by those belonging to subclade C1 (cluster C1-M27: 16 isolates, 20.3%; cluster non-C1-M27: 6 isolates, 7.6%) and clade A (4 isolates; 5.1%). The C2 subclade isolates showed a higher VF-encoding gene score (mean 14.250) compared with the C1-M27 cluster isolates (mean 10.875) (P < 0.001). In conclusion, this study highlights the epidemiological differences between the ESBL-EC isolated from two hospitals of France and Spain obtain in 2015 and reports, for the first time, the presence of clone ST1193 in Spain.

Clonal Structure, Virulence Factor-encoding Genes and Antibiotic Resistance of Escherichia coli, Causing Urinary Tract Infections and Other Extraintestinal Infections in Humans in Spain and France during 2016.

Escherichia coli is the main pathogen responsible for extraintestinal infections. A total of 196 clinical E. coli consecutively isolated during 2016 in Spain (100 from Lucus Augusti hospital in Lugo) and France (96 from Beaujon hospital in Clichy) were characterized. Phylogroups, clonotypes, sequence types (STs), O:H serotypes, virulence factor (VF)-encoding genes and antibiotic resistance were determined. Approximately 10% of the infections were caused by ST131 isolates in both hospitals and approximately 60% of these infections were caused by isolates belonging to only 10 STs (ST10, ST12, ST58, ST69, ST73, ST88, ST95, ST127, ST131, ST141). ST88 isolates were frequent, especially in Spain, while ST141 isolates significantly predominated in France. The 23 ST131 isolates displayed four clonotypes: CH40-30, CH40-41, CH40-22 and CH40-298. Only 13 (6.6%) isolates were carriers of extended-spectrum beta-lactamase (ESBL) enzymes. However, 37.2% of the isolates were multidrug-resistant (MDR). Approximately 40% of the MDR isolates belonged to only four of the dominant clones (B2-CH40-30-ST131, B2-CH40-41-ST131, C-CH4-39-ST88 and D-CH35-27-ST69). Among the remaining MDR isolates, two isolates belonged to B2-CH14-64-ST1193, i.e., the new global emergent MDR clone. Moreover, a hybrid extraintestinal pathogenic E.coli (ExPEC)/enteroaggregative isolate belonging to the A-CH11-54-ST10 clone was identified.

Association Between Kinetics of Early Biofilm Formation and Clonal Lineage in Escherichia coli.

BACKGROUND: Escherichia coli biofilm formation has mostly been assessed in specific pathogenic E. coli groups. Here, we assessed the early biofilm formation (EBF), i.e., adhesion stage, using the BioFilm Ring Test® on 394 E. coli clinical isolates (EC) [196 consecutively isolated (CEC) in 2016 and 198 ESBL-producing E. coli (ESBLEC) isolated in 2015]. Then, biofilm-forming ability was contrasted with phylogroups, clonotypes (fumC-fimH), and sequence types (STs), all being used to define clones, virulence factors (VF), and FimB. RESULT: According to both biofilm production levels at 2, 3, and 5 h, and EBF kinetics over 5 h, CEC and ESBLEC isolates segregated into three EBF groups: strong (G1), moderate (G2), and weak (G3) producers. At 2 h, strong producers were more frequent among CEC (n = 28; 14.3%) than among ESBLEC (n = 8; 4%) (P = 0.0004). As CEC and ESBLEC isolates showed similar individual EBF kinetics in each group, a comparison of isolate features between each group was applied to gathered CEC and ESBLEC isolates after 2 h of incubation, 2 h being the most representative time point of the CEC and ESBLEC isolate segregation into the three groups. Phylogroup B2 displayed by 51.3% of the 394 isolates was more frequent in G1 (77.8%) than in G3 (47.6%) (P = 0.0006). The 394 isolates displayed 153 clones, of which 31 included at least three isolates. B2-CH14-2-ST127, B2-CH40-22-ST131, B2-CH52-5/14-ST141, and E-CH100-96-ST362 clones were associated with G1 (P < 0.03) and accounted for 41.7% of G1 isolates. B2-CH40-30-ST131 clone was associated with G3 (P < 0.0001) and accounted for 25.5% of G3 isolates. VF mean was higher among G1 than among G3 isolates (P < 0.001). FimB-P2 variant was associated with G1 (P = 0.0011) and FimB-P1 variant was associated with G3 (P = 0.0023). Clone, some VF, and FimB were associated with EBF, with clonal lineage being able to explain 72% of the variability of EBF. CONCLUSION: Among our 394 isolates, <10% are able to quickly and persistently produce high biofilm levels over 5 h. These isolates belong to a few clones previously described in various studies as dominant gut colonizers in mammalians and birds and comprised the B2-CH40-22-ST131 clone, i.e., the ancestor of the globally disseminated B2-CH40-30-ST131 clone that is the dominant clone among the weak biofilm producers.

In-vivo loss of carbapenem resistance by extensively drug-resistant Klebsiella pneumoniae during treatment via porin expression modification.

Klebsiella pneumoniae, an Enterobacteriaceae that mostly causes hospital-acquired infections, belongs to the recently published WHO's list of antibiotic-resistant pathogens that pose the greatest threat to human health. Indeed, K. pneumoniae is the enterobacterial species most concerned by both resistance to extended-spectrum cephalosporins, due to extended-spectrum ß-lactamase (ESBL) production, and resistance to carbapenems, i.e. the ß-lactams with the broadest activity. Carbapenem resistance is related not only to carbapenemase production, but also the production of ESBL or AmpC and the loss of general porins. Here, we characterized the mechanisms that deprived a urinary ESBL-producing, porin-deficient K. pneumoniae isolate, isolated 13 days after the end of a 40-day course of imipenem treatment, of its carbapenem resistance. These mechanisms were observed in two in-vivo derivatives of this isolate and consisted of mutations in genes encoding molecules that participate in the downregulation of the synthesis of PhoE, a porin specialized in phosphate transport. We obtained three new derivatives from one of the two original derivatives, following in-vitro antibiotic pressure, in which the carbapenem resistance was restored because of mutations in genes encoding molecules that participate in the upregulation of PhoE synthesis. Thus, we uncovered novel mechanisms of carbapenem resistance/susceptibility switching in K. pneumoniae.

Diversity and functionality of plasmid-borne VagCD toxin-antitoxin systems of Klebsiella pneumoniae.

Objectives: To explore the VagCD toxin-antitoxin (TA) systems encoded on plasmids in multiresistant Klebsiella pneumoniae strains. Methods: Previously sequenced K. pneumoniae plasmids were used for in silico analysis and a collection of 63 resistant K. pneumoniae strains was used for epidemiological study. Functional analysis was done after separate cloning of the toxin gene under the control of the arabinose-inducible promoter of pBAD43 and of the antitoxin gene under the control of the constitutive promoter of pUC19. Results: In silico , two types of VagCD systems, VagCD1 and VagCD2, encoded on K. pneumoniae plasmids could be distinguished, 15% carrying one of these TA systems. Moreover, in a collection of antibiotic-resistant K. pneumoniae strains including ESBL or carbapenemase producers, 17.5% of isolates were found to harbour a VagCD TA system. VagCD1 and VagCD2 were proved functional TA systems, with VagD the toxin and VagC its antitoxin, not only in K. pneumoniae but also in Escherichia coli and other Enterobacteriaceae. Toxin expression was found to induce a significant decrease in a bacterial population resulting from both bactericidal and bacteriostatic effects. Conclusions: The vagCD genes of K. pneumoniae encode a functional broad-spectrum TA system and are conserved on the large multiple antibiotic resistance-conferring plasmids in this species.

Laboratory-based strategy using a new marketed polymerase chain reaction assay to manage diarrheic episodes among patients from rehabilitation and long-term care facilities.

Management of Norovirus and Clostridium difficile gastroenteritis is challenging for rehabilitation and long-term care facilities. We evaluated the contribution of a 2-step laboratory-based strategy, including a new ready-to-use Norovirus polymerase chain reaction assay to promote isolation precautions. C difficile and Norovirus were successively identified from 17% and 23% of 52 episodes of diarrhea, respectively, during the winter season, leading to 100% adequate isolation measures. In patient populations with numerous risk factors for diarrhea, a combined laboratory-based approach could improve infection control.