The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria–Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo .     

Distribution of integron-associated trimethoprim–sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals

Aims: To compare the distribution of integrons and trimethoprim–sulfamethoxazole resistance genes among Escherichia coli isolates from humans and food‐producing animals. Methods and Results: A collection of 174 multidrug‐resistant E. coli isolates obtained from faecal samples of food‐producing animals (n = 64) and humans (n = 59), and patients with urinary tract infections (n = 51) in Hong Kong during 2002–2004 were studied. The strains were analysed for their phylogenetic groups, the presence of sul genes (sul1 and sul2), integrons (intl1 and intl2) and class 1 integron‐associated dfr cassette genes by PCR, restriction enzyme analysis and sequencing. Integrons were identified in 110 (63·2%) isolates. The prevalence of integrons was significantly different according to the specimen sources (animal faecal 84·4%, human faecal 67·8% and human urinary 31·4%) and phylogenetic groups (B1 80·8%, A 77·6%, D 54·1% and B2 11·5%). Faecal isolates (both human and animal) are more likely to belong to group A and B1. In contrast, most urinary isolates were either groups B2 and D. Among dfr containing isolates, dfrA1 and dfrA12 were almost exclusively found in strains of phylogenetic groups A and B1; and were present in animal and human faecal isolates. In contrast, dfrA17 was found in both faecal and urinary isolates and comprised strains from all phylogenetic groups. The sul1 and sul2 genes were equally prevalent among the isolates irrespective of the specimen source and phylogenetic group status. Pulsed‐field gel electrophoresis analysis of isolates with identical cassette genes showed that they were genetically diverse. Conclusions: More animal faecal isolates carry class 1 integrons than human faecal and human urinary isolates, and the distribution of phylogenetic groups is common across animal and human faecal isolates but different from human urinary isolates. Significance and Impact of the Study: Commensal isolates from food‐producing animals are an important reservoir for integrons carrying antibiotic resistance genes.     

Production and characterization of Escherichia coli enterohemolysin and its effects on the structure of erythrocyte membranes

Hemolysins are cell-damaging protein toxins produced by pathogenic bacteria, which are usually released into the extracellular medium. Escherichia coli enterohemolysin is an intracellular toxin produced during the log phase of growth, with a maximal intracellular accumulation in the late log phase. In the present study, we have employed electron microscopy and SDS-PAGE to assess the effects of enterohemolysin on erythocyte membranes from different species. The erythrocyte cell damage began immediately after exposure to enterohemolysin with chemically detectable changes in cell membrane permeability, and the formation of surface lesions which increased rapidly in size. This process resulted in complete cell destruction. Ring-shaped structures with a diameter of 10nm were observed by electron microscopy after treatment of horse erythrocyte membranes with enterohemolysin. The ring structures were found clustered and irregularly distributed on the surface of the membranes. Following incubation of the toxin with horse erythrocyte ghosts and detergent-solubilization, the enterohemolysin was isolated from the cytoplasm in its membrane-bound form by sucrose density gradient. SDS-PAGE and silver staining of deoxycholate-solubilized target membranes revealed heterogeneous forms of the toxin. By using SDS-PAGE and gel filtration, the molecular weight of the toxin was estimated to be 35 kDa. With respect to species specificity, horse erythrocytes showed the highest sensitivity to the enterohemolysin, followed by human and guinea pig erythrocytes. The hemolytic sensitivity correlated with the toxin binding capacity of erythrocyte membranes of different animal species. The degree of hemolysis was unaffected by temperature in the range of 4 degrees C-37 degrees C and was optimal at pH 9.0. In contrast to pore-forming cytolysins, the hemolytic activity of enterohemolysin was enhanced continuously in the presence of increasing concentrations of dextran 4 and dextran 8 within the range of 5 to 30 mM. Trypsin sensitivity of membrane-bound enterohemolysin indicates that the cell surface is the most likely target site for this toxin. Additionally, the fact that proteinase and phosphatase inhibitors failed to inhibit lysis suggests that enterohemolysin alters and disrupts cell membranes by a detergent-like mechanism.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Characterization of antimicrobial resistant Escherichia coli isolated from processed bison carcasses

AIM: To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. METHODS AND MATERIALS: The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. RESULTS: Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16.7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13.0%), sulfamethoxazole (7.9%) and streptomycin (5.8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. CONCLUSION: This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.     

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.     

Phenotypic and molecular characteristics of Escherichia coli isolated from aquatic environment of Bangladesh

Pathogenic Escherichia coli remains important etiological agent of infantile diarrhea in Bangladesh. Previous studies have focused mostly on clinical strains, but very little is known about their presence in aquatic environments. The present study was designed to characterize potentially pathogenic E. coli isolated between November 2001 and December 2003 from aquatic environments of 13 districts of Bangladesh. Serotyping of 96 randomly selected strains revealed O161 to be the predominant serotype (19%), followed by O55 and O44 (12% each), and 11% untypable. Serotype-based pathotyping of the E. coli strains revealed 47%, 30%, and 6% to belong to EPEC, ETEC, and EHEC pathotypes, respectively. The majority of the 160 strains tested were resistant to commonly used antimicrobial agents. Plasmid pro-filing showed a total of 17 different bands ranging from 1.3 to 40 kb. However, 35% of the strains did not contain any detectable plasmid, implying no correlation between plasmid and drug resistance. Although virulence gene profiling revealed 97 (61%) of the strains to harbor the gene encoding heat-stable enterotoxin (ST), 2 for the gene encoding Shiga toxin (Stx), and none for the gene for heat-labile enterotoxin (LT), serotype-based pathotyping of E. coli was not fully supported by this gene profiling. A dendrogram derived from the PFGE patterns of 22 strains of three predominant serogroups indicated two major clusters, one containing mainly serogroup O55 and the other O8. Three strains of identical PFGE profiles belonging to serogroup O55 were isolated from three distinct areas, which may be of epidemiological significance. Finally, it may be concluded that serotype-based pathotyping may be useful for E. coli strains of clinical origin; however, it is not precise enough for reliably identifying environmental strains as diarrheagenic.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

Extended-spectrum beta-lactamase-producing Escherichia coli harbouring sul and mcr-1 genes isolates from fish gut contents in the Mekong Delta,Vietnam

This study investigated the existence of sulfonamides and colistin resistance genes among extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli recovered from fish gut in Vietnam and evaluated the susceptibility patterns of the ESBL-producing E. coli to relevant antimicrobials. A total of 88 ESBL-producing E. coli isolates were analysed for the presence of the ESBLs, sul (1, 2, 3) and mcr (1–3) genes by PCR. Antimicrobial resistance phenotypes of isolates were determined by disc diffusion. Results showed that: (i) A high prevalence of 94·3% of sulfonamide resistance was observed in 88 isolates. Moreover, the existence of 2·3% of ESBL-producing E. coli harbouring mcr-1 gene were detected; (ii) The phylogenetic types A and B1 were most frequent, and the bla_CTX-M group1 and bla_TEM genes encoding ESBL were detected in 47·7% of the isolates; (iii) ESBL-producing E. coli harbouring mcr-1 gene exhibited resistance to 11 antibiotics. The existence of mcr-1 and sul1,2,3 genes and the extremely high level of multiple drug resistance in all ESBL-producing E. coli isolates obtained from sampled fish in Vietnam is a major concern. Therefore, it is imperative to monitor ESBL-producing E. coli in the river waters of Vietnam.     

Antimicrobial resistance of Escherichia coli isolated from freshwaters and hospital effluents in Belgium

The purpose of this work was to evaluate the level of antimicrobial resistant Escherichia coli isolates in freshwaters and hospital effluents in Belgium. The samples were collected from 24 locations along the Ourthe, Vesdre, Amblève and Meuse rivers and in the wastewater effluents of several hospitals. The sampling stations in rivers were classified according to the dominant land covers of the rivers (rural, urban and forest areas). Two sampling campaigns were organized in May and October 2019 to highlight a possible seasonal effect. A total of 938 E. coli strains were isolated on Chromogenic Selective Tryptone Bile X-glucuronide (TBX) and TBX supplemented with amoxicillin (TBX+AMX) media. Disk diffusion assays were performed following the EUCAST's recommendations to assess the antimicrobial resistance against 12 antibiotics. A total of 32·7% of strains were at least resistant to one antibiotic and 24·6% were multiple antimicrobial resistant strains on TBX. The highest resistance rates were found for ampicillin (AMP), amoxicillin coupled with clavulanic acid (AMC) and sulfamethoxazole/trimethoprim (SXT). The lowest resistance rates were observed for meropenem (MEM) and ertapenem (ETP), which are last resort antibiotics. No significant difference was observed between both campaigns for the resistance rate to antibiotics.     

Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City

Aims:  To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli .
Methods and Results:  We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain.
Conclusions:  Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs.
Significance and Impact of the Study:  The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c . 8 μg ml⁻¹) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.