Repeated Isolation of Extended-Spectrum-ß-Lactamase-Positive Escherichia coli Sequence Types 648 and 131 from Community Wastewater Indicates that Sewage Systems Are Important Sources of Emerging Clones of Antibiotic-Resistant Bacteria.

Antibiotic resistance in bacteria is an emerging problem globally. Resistant bacteria are found in human and animal microbiota, as well as in the environment. Wastewater receives bacteria from all these sources and thus can provide a measurement of abundance and diversity of antibiotic-resistant bacteria circulating in communities. In this study, water samples were collected from a wastewater pump station in a Norwegian suburban community over a period of 15 months. A total of 45 daily samples were cultured and analyzed for the presence of Escherichia coli Eighty E. coli-like colonies were collected from each daily sample and then phenotyped and analyzed for antibiotic resistance using the PhenePlate-AREB system. During the sampling period, two unique E. coli phenotypes with resistance to cefotaxime and cefpodoxime indicating carriage of extended-spectrum ß-lactamases (ESBL) were observed repeatedly. Whole-genome sequencing of 15 representative isolates from the two phenotypes identified these as two distinct clones belonging to the two globally spread E. coli multilocus sequence types (STs) ST131 and ST648 and carrying blaCTX-M-15 The number of ESBL-positive E. coli strains in the community wastewater pump station was 314 of 3,123 (10%) analyzed E. coli strains. Of the ESBL-positive isolates, 37% belonged to ST648, and 7% belonged to ST131. Repeated findings of CTX-M-15-positive ST648 and ST131 over time indicate that these STs are resident in the analyzed wastewater systems and/or circulate abundantly in the community.

Persistence of Lactobacilli in Postmenopausal Women - A Double-Blind, Randomized, Pilot Study.

AIM: To investigate the ability of lactobacilli to persist in the genital area (vagina and labia) of women after the topical application of an ointment containing Lactobacillus gasseri LN40, L. fermentum LN99 and L. rhamnosus LN113. Secondary objectives were to study the presence of Escherichia coli and other contaminants, as well as subjective symptoms in the genital tract. METHODS: Eighteen healthy postmenopausal women were randomized to use either the study product or placebo for 10 days. Gynecological examinations, labial and vaginal samplings for bacterial cultivation were performed at baseline (visit 1), after treatment (visit 2), and at a 10-day follow-up (visit 3). LN strains were identified by specific cultivation methods. Subjective symptoms were evaluated by a self-administered questionnaire. RESULTS: The presence of LN99 was shown in 7 out of 8 women in the investigational group at visit 2 (p < 0.001 compared to placebo) and in 5 out of 8 at visit 3 (p < 0.05), whereas the presence of LN113 was shown in 2 out of 8 at visit 2 and in 1 out of 8 at visit 3. Subjective symptoms were significantly reduced (p < 0.01) at visits 2 and 3 for both products. CONCLUSION: Topical application of a probiotic ointment is feasible to achieve persistence of lactobacilli for at least 10 days.

Circulation of enterotoxigenic Escherichia coli (ETEC) isolates expressing CS23 from the environment to clinical settings.

IMPORTANCE: The importance of clean water cannot be overstated. It is a vital resource for maintaining health and well-being. Unfortunately, water sources contaminated with fecal discharges from animal and human origin due to a lack of wastewater management pose a significant risk to communities, as they can become a means of transmission of pathogenic bacteria like enterotoxigenic E. coli (ETEC). ETEC is frequently found in polluted water in countries with a high prevalence of diarrheal diseases, such as Bolivia. This study provides novel insights into the circulation of ETEC between diarrheal cases and polluted water sources in areas with high rates of diarrheal disease. These findings highlight the Choqueyapu River as a potential reservoir for emerging pathogens carrying antibiotic-resistance genes, making it a crucial area for monitoring and intervention. Furthermore, the results demonstrate the feasibility of a low-cost, high-throughput method for tracking bacterial pathogens in low- and middle-income countries, making it a valuable tool for One Health monitoring efforts.

Escherichia coli ST2797 Is Abundant in Wastewater and Might Be a Novel Emerging Extended-Spectrum Beta-Lactamase E. coli.

The increasing prevalence of antibiotic-resistant bacteria is an emerging threat to global health. The analysis of antibiotic-resistant enterobacteria in wastewater can indicate the prevalence and spread of certain clonal groups of multiresistant bacteria. In a previous study of Escherichia coli that were isolated from a pump station in Norway over 15 months, we found a recurring E. coli clone that was resistant to trimethoprim, ampicillin, and tetracycline in 201 of 3,123 analyzed isolates (6.1%). 11 representative isolates were subjected to whole-genome sequencing and were found to belong to the MLST ST2797 E. coli clone with plasmids carrying resistance genes, including blaTEM-1B, sul2, dfrA7, and tetB. A phenotypic comparison of the ST2797 isolates with the uropathogenic ST131 and ST648 that were repeatedly identified in the same wastewater samples revealed that the ST2797 isolates exhibited a comparable capacity for temporal survival in wastewater, greater biofilm formation, and similar potential for the colonization of mammalian epithelial cells. ST2797 has been isolated from humans and has been found to carry extended spectrum ß-lactamase (ESBL) genes in other studies, suggesting that this clonal type is an emerging ESBL E. coli. Collectively, these findings show that ST2797 was more ubiquitous in the studied wastewater than were the infamous ST131 and ST648 and that ST2797 may have similar abilities to survive in the environment and cause infections in humans. IMPORTANCE The incidence of drug-resistant bacteria found in the environment is increasing together with the levels of antibiotic-resistant bacteria that cause infections. The COVID-19 pandemic has shed new light on the importance of monitoring emerging threats and finding early warning systems. Therefore, to mitigate the antimicrobial resistance burden, the monitoring and early identification of antibiotic-resistant bacteria in hot spots, such as wastewater treatment plants, are required to combat the occurrence and spread of antibiotic-resistant bacteria. Here, we applied a PhenePlate system as a phenotypic screening method for genomic surveillance and discovered a dominant and persistent E. coli clone ST2797 with a multidrug resistance pattern and equivalent phenotypic characteristics to those of the major pandemic lineages, namely, ST131 and ST648, which frequently carry ESBL genes. This study highlights the continuous surveillance and report of multidrug resistant bacteria with the potential to spread in One Health settings.

Diversity and antibiotic resistance among Escherichia coli populations in hospital and community wastewater compared to wastewater at the receiving urban treatment plant.

Bacterial diversity and antimicrobial resistance patterns among the indicator organism Escherichia coli were monitored in wastewater samples collected over one year from a hospital (HW), a community (CW) and the receiving urban (UW) wastewater treatment plant (WWTP). We compared levels of antibiotic resistance in the different types of wastewater, and identified whether resistant strains were endemic in the wastewater system. If so, implementation of local treatment at certain resistance hotspots (e.g. hospital outlets) could be used to decrease the amount of resistant bacteria in the wastewater. E. coli from HW (n = 2644), CW (n = 2525) and UW (n = 2693) were analyzed by biochemical phenotyping (PhenePlate System) and antimicrobial susceptibility testing to nine antibiotics (AREB System). The phenotypic diversities of the total E. coli populations were similar for all three sites (Simpson's Diversity index, Di = 0.973), however for individual samples, HW showed low diversities (Median Di = 0.800) and the E. coli flora was often dominated by strains that may have originated from the fecal flora of single individuals. The diversities in CW samples was higher (Median Di = 0.936), and UW samples showed similar diversities as the whole collection of isolates (Median Di = 0.971). Resistance to at least one of the nine antibiotics was observed in 45% of the HW isolates, 44% of CW isolates, and 33% of UW isolates. Resistance to gentamicin and chloramphenicol was uncommon (3.2 and 5.3%, respectively), whereas resistance to tetracycline and ampicillin was most common (24% and 31%, respectively). Extended-spectrum beta-lactamase-producing E. coli (ESBL-EC) were more common in HW (11.5%) and in CW (6.9%) compared to UW (3.7%). A high diversity (Di = 0.974) was observed among ESBL-EC isolates from UW (n = 99), indicating absence of any clonal structure among these isolates. Common PhP types of ESBL-EC often dominated in each HW sample, but were not identified across different samples, whereas ESBL-EC in CW showed low diversity (Di = 0.857) and were dominated by a specific PhP type that was found across almost all CW samples. The antibiotic resistance rates were highest in hospital wastewater, but surprisingly they were also high in the studied community wastewater, compared to the urban wastewater. The relative contribution of HW seemed low in terms of dissemination of antibiotic resistant bacteria to the WWTP.

Surveillance of antimicrobial resistance among Escherichia coli in wastewater in Stockholm during 1 year: does it reflect the resistance trends in the society?

The resistance patterns of Escherichia coli in untreated (raw) urban wastewater (UW) was monitored by repeated sampling during 1 year. Comparison with data from wastewater samples collected from hospital wastewater (HW) in the same urban area was made. A total of 1326 E. coli isolates from 17 UW samples and 451 isolates from six HW samples were analysed by typing using the PhenePlate™ system, and their susceptibility towards 10 antibiotics was determined. Resistance to at least one antibiotic was observed in 34% of the UW isolates and 55% of the HW isolates. For UW isolates, phenotypic diversity was lower among antibiotic-susceptible than among antibiotic-resistant isolates, indicating a higher presence of clonal groups among susceptible isolates. Total antibiotic resistance measured as the Multiple Antibiotic Resistance (MAR) index was 0.08 for UW compared with 0.19 for HW, and increased over time for UW isolates, indicating increasing resistance among E. coli in the urban population during the studied time period. Resistance to all included ß-lactam antibiotics was detected in 2.4% of UW isolates and 14.0% of HW isolates, and 73/75 (97%) analysed isolates were confirmed to be extended-spectrum ß-lactamase (including plasmid-mediated AmpC)-producing E. coli. Thus, by cultivating samples from wastewater and analysing many independent isolates per sample, increasing frequencies of antibiotic resistance in UW were detected during 1 year that may reflect increasing faecal carriage of resistant bacteria in the society. Surveillance of antibiotic resistance in wastewater could be a valuable tool for screening of resistance trends on a population level.

Diversity of intestinal Escherichia coli populations in Nicaraguan children with and without diarrhoea.

Escherichia coli remains an important aetiological agent of infantile diarrhoea in Nicaragua. However, little is known about whether there is a high prevalence of endemic strains or whether infection is due to the epidemic spread of virulent clones. This study was undertaken to determine the diversity and distribution of clonal groups in a population of intestinal E. coli isolated from the faeces of children from León, Nicaragua, with (n=381) and without (n=145) diarrhoea, between March 2005 and September 2006. All samples had been screened previously for the presence of diarrhoeagenic E. coli (DEC) markers by multiplex PCR. From each sample, 8 E. coli colonies (where available) were analysed by biochemical fingerprinting (PhP-RE system), yielding a total of 4009 tested isolates. On average, three different biochemical phenotypes (BPTs) were found among the eight colonies analysed from each sample. The total diversity, measured as Simpson's diversity index (Di), was 0.97 among all 4009 isolates studied. Cluster analysis of data from all 4009 isolates revealed 24 common BPTs (identified in at least 1 % of the isolates) and 234 less common BPTs. Similar Di values were obtained among isolates from infants with and without diarrhoea, indicating that no widespread outbreak of DEC had occurred. Moreover, among samples that were positive for the DEC types enteroaggregative E. coli, enteropathogenic E. coli and enterotoxigenic E. coli (ETEC) carrying the eltB gene, the diversities were almost as high as among non-DEC samples, whereas samples positive for ETEC carrying estA, enteroinvasive E. coli and enterohaemorrhagic E. coli showed lower diversities, indicating the prevalence of virulent clonal groups among these samples. The PhenePlate patterns of the 24 common BPTs identified here were compared with those obtained from E. coli isolated in a cohort infant study performed in 1991-1992 in the same area. Only 4 % of the isolates from the 1990s were similar to any of the common BPTs found in the present study.