Background. The determinants of the spread of extended-spectrum β-lactamase–producing Escherichia coli (ESBLEC) in the community remain unclear. To evaluate its dissemination in the environment, we analyzed the ESBLEC population throughout an urban wastewater network. Methods. Samples were collected weekly, over a 10-week period, from 11 sites throughout the wastewater network of Besançon city (France). Total E. coli and ESBLEC loads were determined for each sample. As a control, we analyzed 51 clinical ESBLEC isolates collected at our hospital. We genotyped both environmental and clinical ESBLEC by pulsed-field gel electrophoresis and multilocus sequence typing and identified their bla_ESBL genes by sequencing. Results. The E. coli load was higher in urban wastewater than in hospital wastewater (7.5 × 10⁵ vs 3.5 × 10⁵ CFU/mL, respectively). ESBLEC was recovered from almost all the environmental samples and accounted for 0.3% of total E. coli in the untreated water upstream from the wastewater treatment plant (WWTP). The ESBLEC load was higher in hospital wastewater than in community wastewater (27 × 10³ vs 0.8 × 10³ CFU/mL, respectively). Treatment by the WWTP eliminated 98% and 94% of total E. coli and ESBLEC, respectively. The genotyping revealed considerable diversity within both environmental and clinical ESBLEC and the overrepresentation of some clonal complexes. Most of the sequence types displayed by the clinical isolates were also found in the environment. CTX-M enzymes were the most common enzymes whatever the origin of the isolates. Conclusions. The treatment at the WWTP led to the relative enrichment of ESBLEC. We estimated that >600 billion of ESBLEC are released into the river Doubs daily and the sludge produced by the WWTP, used as fertilizer, contains 2.6 × 10⁵ ESBLEC per gram.

In 2006, 0.6% of healthy subjects living in the Paris area had extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in their gut. To assess the evolution of this rate, a study identical to that of 2006 was conducted in 2011. Healthy adults who visited the IPC check-up centre in February–March 2011 and agreed to participate, provided stools and answered a questionnaire on the visit day. Stools were analysed to detect ESBL producers and to isolate the dominant E. coli population. ESBLs were molecularly characterized. For the subjects harbouring ESBL-producing E. coli, the phylogenetic group and sequence type (ST) were determined for both ESBL-producing and dominant E. coli isolates. PFGE profiles were also determined when two types of isolates had the same ST. Among the 345 subjects included, 21 (6%) had ESBL-producing E. coli faecal carriage. None of the previously published risk factors was identified. CTX-M accounted for 86% and SHV-12 for 14%. Dominant and ESBL-producing E. coli were similarly distributed into phylogenetic groups (A, 52%–48%; B1, 5%; B2, 24%–14%; and D, 19%–33%). Dominant and ESBL-producing E. coli displayed a polyclonal structure (18 STs each). However, ST10 and ST131 were identified in dominant and ESBL-producing E. coli isolates from different subjects. Most (20/21) ESBL producers were subdominant and belonged (16/21) to STs different from that of the corresponding dominant E. coli. The 10-fold increase in the rate of healthy subjects with ESBL-producing E. coli faecal carriage over a 5 year period suggests wide dissemination of these isolates in the Parisian community.

Bacterial resistance to β-lactams may rely on acquired β-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette bla_OXA-28 encoding an extended spectrum β-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the β-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the β-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria. The bacterial SOS response is a conserved regulatory network that is induced in response to DNA damage. Its activation in vitro leads to the emergence of resistance to antibiotics, leading to speculation that this is an important clinical mechanism of acquiring resistance. We found evidence here that antibiotic-induced SOS response plays a role in bacterial genome rearrangement in vivo within humans. The major classes of antibiotics can trigger the bacterial SOS response and our data raise questions about their wide use and their subsequent effect on the bacterial genetic adaptability. This suggests that emergence of antibiotic resistance during therapy could be reduced by inhibiting the bacterial SOS response. We showed that acquired resistance genes could spread latently in susceptible bacterial strains until needed. These findings could impact current policies for control of antibiotic resistance, which rely on the detection of resistant bacteria and on the assumption that resistance mechanisms have a functional cost to the bacteria. More generally, SOS response may spur changes in the behavior of bacteria and their faster adaptation to hostile environments, including humans.

The Pseudomonas aeruginosa-containing wastewater released by hospitals is treated by wastewater treatment plants (WWTPs), generating sludge, which is used as a fertilizer, and effluent, which is discharged into rivers. We evaluated the risk of dissemination of antibiotic-resistant P. aeruginosa (AR-PA) from the hospital to the environment via the wastewater network. Over a 10-week period, we sampled weekly 11 points (hospital and urban wastewater, untreated and treated water, sludge) of the wastewater network and the river upstream and downstream of the WWTP of a city in eastern France. We quantified the P. aeruginosa load by colony counting. We determined the susceptibility to 16 antibiotics of 225 isolates, which we sorted into three categories (wild-type, antibiotic-resistant and multidrug-resistant). Extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) were identified by gene sequencing. All non-wild-type isolates (n = 56) and a similar number of wild-type isolates (n = 54) were genotyped by pulsed-field gel electrophoresis and multilocus sequence typing. Almost all the samples (105/110, 95.5%) contained P. aeruginosa, with high loads in hospital wastewater and sludge (≥3×10⁶ CFU/l or/kg). Most of the multidrug-resistant isolates belonged to ST235, CC111 and ST395. They were found in hospital wastewater and some produced ESBLs such as PER-1 and MBLs such as IMP-29. The WWTP greatly reduced P. aeruginosa counts in effluent, but the P. aeruginosa load in the river was nonetheless higher downstream than upstream from the WWTP. We conclude that the antibiotic-resistant P. aeruginosa released by hospitals is found in the water downstream from the WWTP and in sludge, constituting a potential risk of environmental contamination.

This study aimed to determine the genetic diversity of clinical multidrug-resistant Pseudomonas aeruginosa . We used pulsed-field gel electrophoresis and multilocus sequence typing to analyze 187 strains isolated in different French hospitals. To illustrate the diversity of resistance mechanisms to antibiotics in a given clone, we identified β-lactamases with an extended spectrum by using phenotypic and genotypic methods. Typing results showed that the majority of our multidrug-resistant isolates belong to a few clonal types (ST235, ST111, and ST175) that are already spreading worldwide. These successful international clones sporadically produced extended-spectrum β-lactamase-encoding genes but mostly became extensively resistant to β-lactams after derepression of intrinsic resistance mechanisms (i.e., AmpC cephalosporinase). Our results indicate that cross-transmission plays a major role in the spread of multidrug-resistant P. aeruginosa in hospital settings.