ABSTRACT
Urinary tract infections (UTIs) represent a clinical and epidemiological problem of worldwide impact that affects the economy and the emotional state of the patient. Control of the condition is complicated due to multidrug resistance of pathogens associated with the disease. Considering the difficulty in carrying out effective treatment with antimicrobials, it is necessary to propose alternatives that improve the clinical status of the patients. With this purpose, in a previous study, the safety and immunostimulant capacity of a polyvalent lysate designated UNAM-HIMFG prepared with different bacteria isolated during a prospective study of chronic urinary tract infection (CUTI) was evaluated. In this work, using an animal model, results are presented on the immunostimulant and protective activity of the polyvalent UNAM-HIMFG lysate to define its potential use in the control and treatment of CUTI. Female Balb/c mice were infected through the urethra with Escherichia coli CFT073 (UPEC O6:K2:H1) strain; urine samples were collected before the infection and every week for up to 60 days. Once the animals were colonized, sublingual doses of UNAM-HIMFG lysate were administrated. The colonization of the bladder and kidneys was evaluated by culture, and their alterations were assessed using histopathological analysis. On the other hand, the immunostimulant activity of the compound was analyzed by qPCR of spleen mRNA. Uninfected animals receiving UNAM-HIMFG lysate and infected animals administered with the physiological saline solution were used as controls. During this study, the clinical status and evolution of the animals were evaluated. At ninety-six hours after infection, the presence of CFT073 was identified in the urine of infected animals, and then, sublingual administration of UNAM-HIMFG lysate was started every week for 60 days. The urine culture of mice treated with UNAM-HIMFG lysate showed the presence of bacteria for three weeks post-treatment; in contrast, in the untreated animals, positive cultures were observed until the 60th day of this study. The histological analysis of bladder samples from untreated animals showed the presence of chronic inflammation and bacteria in the submucosa, while tissues from mice treated with UNAM-HIMFG lysate did not show alterations. The same analysis of kidney samples of the two groups (treated and untreated) did not present alterations. Immunostimulant activity assays of UNAM-HIMFG lysate showed overexpression of TNF-α and IL-10. Results suggest that the lysate activates the expression of cytokines that inhibit the growth of inoculated bacteria and control the inflammation responsible for tissue damage. In conclusion, UNAM-HIMFG lysate is effective for the treatment and control of CUTIs without the use of antimicrobials.
Subject(s)
Escherichia coli Infections , Mice, Inbred BALB C , Urinary Bladder , Urinary Tract Infections , Uropathogenic Escherichia coli , Animals , Urinary Tract Infections/microbiology , Urinary Tract Infections/immunology , Female , Mice , Urinary Bladder/microbiology , Urinary Bladder/immunology , Urinary Bladder/pathology , Urinary Bladder/drug effects , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Uropathogenic Escherichia coli/immunology , Uropathogenic Escherichia coli/pathogenicity , Disease Models, Animal , Adjuvants, Immunologic/pharmacology , Bacterial LysatesABSTRACT
Worldwide, Urinary Tract Infections (UTIs) are an important health problem with many cases reported annually, women being the most affected. UTIs are relevant because they can become a recurrent condition, associated with different factors that contribute to the chronicity of the disease (cUTI). cUTI can be classified as persistent (peUTI) when the causative agent is the same each time the infection occurs or as reinfection (reUTI) when the associated microorganism is different. The purpose of this work was to characterize Escherichia coli isolates obtained in two prospective studies of patients with cUTI, to define which of them corresponded to peUTI and which to reUTI. A total of 394 isolates of E. coli were analyzed by agglutination with specific sera, antimicrobial susceptibility by diffusion disc test, and the phylogroups and presence of genes associated with virulence by PCR assays. Additionally, in some characterized strains adherence, invasiveness, and biofilm formation were analyzed by in vitro assays. The results showed that the peUTI strains belonged mainly to the classical UPEC serogroups (O25, O75, O6), were included in the B2 phylogroup, carried a great number of virulence genes, and were adherent, invasive, and biofilm-forming. Meanwhile, reUTI strains showed great diversity of serogroups, belonged mainly in the A phylogroup, and carried fewer virulence genes. Both peUTI and reUTI strains showed extensively drug-resistant (XDR) and multidrug-resistant (MDR) profiles in the antimicrobial susceptibility test. In conclusion, it appears that peUTIs are caused principally by classical UPEC strains, while reUTIs are caused by strains that appear to be a part of the common E. coli intestinal biota. Moreover, although both peUTI and reUTI strains presented different serotypes and phylogroups, their antimicrobial resistance profile (XDR and MDR) was similar, confirming the importance of regulating prophylactic treatments and seeking alternatives for the treatment and control of cUTI. Finally, it was possible to establish the features of the E. coli strains responsible for peUTI and reUTI which could be helpful to develop a fast diagnostic methodology.
Subject(s)
Anti-Infective Agents , Escherichia coli Infections , Urinary Tract Infections , Humans , Female , Escherichia coli/genetics , Follow-Up Studies , Escherichia coli Infections/diagnosis , Prospective Studies , Virulence Factors/analysis , Virulence Factors/genetics , Urinary Tract Infections/diagnosisABSTRACT
One hundred and five uropathogenic Escherichia coli (UPEC) strains from patients with community-acquired urinary tract infections were characterized according to phylogenetic group, virulence factors, serogroup, antibiotic resistance, and genotype. The pathogenic phylogenetic groups (B2, D, and F) were found in 71.4% of the tested strains. Among them, the main uropathogenic serogroups were O8, O25, and O75, in which 97.1% of the strains had a multidrug-resistant profile. Sixteen virulence genes were analysed using a combination of polymerase chain reaction (PCR) assays, with the fimH, irp-2, iutA, aer, iucC, PAI, sat, iroN, usp, and cnf1 genes being mainly found in pathogenic phylogroups. The E. coli O25b-ST131 clone was identified in 32% of the strains assigned to the pathogenic phylogroup B2. These findings demonstrate that virulence genes encoding adhesin components, iron-acquisition systems, toxins, and pathogenicity-associated islands were highly prevalent among the pathogenic phylogroup of UPEC strains.
Subject(s)
Community-Acquired Infections , Escherichia coli Infections , Urinary Tract Infections , Uropathogenic Escherichia coli , Community-Acquired Infections/epidemiology , Escherichia coli Infections/epidemiology , Humans , Iron , Mexico/epidemiology , Phylogeny , Urinary Tract Infections/epidemiology , Uropathogenic Escherichia coli/genetics , Virulence Factors/analysis , Virulence Factors/geneticsABSTRACT
This study aimed to identify and characterize integrons among multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) from outpatients in Mexico City, Mexico. PCR assays were used to screen for the presence of class 1, 2 and 3 integrons, whose PCR products were sequenced to identify the inserted gene cassettes within the variable regions. Out of 83 tested strains, 53 (63.9%) were positive for the presence of class 1 integrons, whereas no integrons were detected in the remaining strains, regardless of their classes. Most of the strains carrying the intI1 gene belonged to the extraintestinal B2 (41.5%) and commensal A (32.1%) phylogroups, and to a lesser extent, the extraintestinal D (20.8%) and commensal B1 (5.7%) phylogroups. Moreover, 8 different gene cassette arrangements were detected, with dfrA17 and aadA5 being the most common (32.1% of the class 1 integron-positive strains), which confer resistance to trimethoprim/sulfamethoxazole and aminoglycosides, respectively. Our results suggest that class 1 integrons are widely distributed among MDR-UPEC strains in Mexico, which may directly or indirectly contribute to the selection of MDR strains. These findings are important for a better understanding of the factors and mechanisms that promote multidrug resistance among UPEC strains.
Subject(s)
Escherichia coli Infections , Uropathogenic Escherichia coli , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Humans , Integrons/genetics , Mexico , Uropathogenic Escherichia coli/geneticsABSTRACT
Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC), whose impact can be exacerbated by multidrug-resistant (MDR) strains. Effective control strategies are, therefore, urgently needed. Among them, phage therapy represents a suitable alternative. Here, we describe the isolation and characterization of novel phages from wastewater samples, as well as their lytic activity against biofilm and adherence of UPEC to HEp-2 cells. The results demonstrated that phage vB_EcoM-phiEc1 (ÏEc1) belongs to Myoviridae family, whereas vB_EcoS-phiEc3 (ÏEc3) and vB_EcoS-phiEc4 (ÏEc4) belong to Siphoviridae family. Phages showed lytic activity against UPEC and gut commensal strains. Phage ÏEc1 lysed UPEC serogroups, whereas phages ÏEc3 and ÏEc4 lysed only UTI strains with higher prevalence toward the O25 serogroup. Moreover, phages ÏEc1 and ÏEc3 decreased both biofilm formation and adherence, whereas ÏEc4 was able to decrease adherence but not biofilm formation. In conclusion, these novel phages showed the ability to decrease biofilm and bacterial adherence, making them promising candidates for effective adjuvant treatment against UTIs caused by MDR UPEC strains. KEY POINTS: Phage with lytic activity against MDR UPEC strains were isolated and characterized under in vitro conditions. A novel method was proposed to evaluate phage activity against bacterial adherence in HEp-2 cell.. Phages represent a suitable strategy to control infections caused by MDR bacteria.
Subject(s)
Bacteriophages , Escherichia coli Infections , Phage Therapy , Urinary Tract Infections , Uropathogenic Escherichia coli , Escherichia coli Infections/therapy , Humans , Urinary Tract Infections/therapyABSTRACT
The O25-ST131 clone was identified within 169 uropathogenic Escherichia coli (UPEC) strains. The 44.8% of the 29 O25-ST131 clones detected were positive to least to one extended-spectrum ß-lactamase gene. The phylogroup D was mainly found. The O25-ST131 clone appeared to be associated with community-acquired UTI in Mexico City.
Subject(s)
Community-Acquired Infections/microbiology , Genotype , Serogroup , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/classification , Uropathogenic Escherichia coli/isolation & purification , Adolescent , Adult , Aged , Community-Acquired Infections/epidemiology , Humans , Mexico/epidemiology , Middle Aged , Urinary Tract Infections/epidemiology , Young Adult , beta-Lactamases/geneticsABSTRACT
INTRODUCTION: Escherichia coli is the major causative agent of urinary tract infections (UTI), and virulence factors are responsible for the severity of these emerging infections. The aim of this study was to evaluate the relationship between virulence determinants and antibiotic susceptibility with phylogenetic groups of E.coli isolates of UTI in two locations in Mexico. METHODS: An analysis was performed on 50 isolates of E.coli from the centre of the country and 57 from a town in the southwest. The isolates were characterized by phenotype (serotyping assays, in vitro adhesion, biofilm formation, production of haemolysin, and antibiotic susceptibility) and genotype (phylogenetic groups and virulence genes). RESULTS: In the centre of the country location the phylogenetic group B2 (60%) and F (12%) were significantly more prevalent and had a higher frequency of genes, fimH (96%), iutA (66%), sat (36%), compared to the southwest location, where the group A (35%) and B1 (21%) were significantly predominant and had fewer virulence genes. About one-fifth (21.5%) of all isolates belonged to the O25-ST131 group. Haemolysin and biofilm producing strains were significantly higher in the southwest location. Resistance to ampicillin (92.5%), tetracycline (76.6%), and trimethoprim/sulfamethoxazole (70.1%) were the most common in both groups. CONCLUSION: The phylogenetic group, virulence factors, and antibiotic susceptibility of the E.coli that causes UTI in the community, varies significantly among the Mexican populations studied. Phylogenetic groups A and B1 may be multidrug resistant and have the ability to produce UTI.
Subject(s)
Uropathogenic Escherichia coli , Drug Resistance, Bacterial , Humans , Mexico , Phylogeny , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/classification , Uropathogenic Escherichia coli/drug effects , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/pathogenicity , Virulence , Virulence FactorsABSTRACT
Escherichia coli clonal group A (CGA) causes urinary tract and other extra-intestinal infections in humans. CGA is an important cause of trimethoprim/sulfamethoxazole (SXT) resistance in extra-intestinal pathogens. We examined the extent to which resistance in this area is related to CGA dissemination of E. coli from urinary tract infections (UTIs) in Mexico City. The virulence backgrounds of the isolates were also characterized. In this study, the frequency of resistance to SXT used for UTI treatment was high (56-65 %), and CGA isolates accounted for 9 of the 78 SXT-resistant isolates (11.5 %). Although all CGA isolates were found to be multidrug resistant (MDR), none of them were extended-spectrum ß-lactamase-producing organisms. The prevalence of CGA among the 45 MDR isolates that we identified was 20 %, indicating that this clonal group moderately contributes to the antibiotic resistance of uropathogenic E. coli isolates in this region. Most of the nine CGA isolates carried transferable, large-size plasmids of approximately 80 to 100 kb, which were able to transfer antimicrobial resistance to E. coli J53 in mating assays. CGA isolates mainly belonged to phylogenetic groups F and D. We found no association between antimicrobial resistance and virulence-associated genes: the median virulence scores of CGA isolates were slightly higher (4.6) than those of non-CGA isolates, whether they were susceptible (3.7) or resistant (3.5) to SXT. Our results indicate that CGA is not a major contributor to the high level of resistance to SXT in this region but, instead, seems to be an important constituent of MDR isolates from UTIs.
Subject(s)
Escherichia coli Infections/microbiology , Urinary Tract Infections/microbiology , Uropathogenic Escherichia coli/genetics , Uropathogenic Escherichia coli/pathogenicity , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Escherichia coli Infections/epidemiology , Genotype , Humans , Mexico/epidemiology , Phylogeny , Trimethoprim, Sulfamethoxazole Drug Combination/pharmacology , Urinary Tract Infections/epidemiology , Uropathogenic Escherichia coli/drug effects , Uropathogenic Escherichia coli/isolation & purification , Virulence Factors/genetics , beta-Lactamases/pharmacologyABSTRACT
Shiga-like toxins (Stx) represent a group of bacterial toxins involved in human and animal diseases. Stx is produced by enterohemorrhagic Escherichia coli, Shigella dysenteriae type 1, Citrobacter freundii, and Aeromonas spp.; Stx is an important cause of bloody diarrhea and hemolytic uremic syndrome (HUS). The aim of this study was to identify the stx1/stx2 genes in clinical strains and outer membrane vesicles (OMVs) of Aeromonas spp., 66 strains were isolated from children who live in Mexico City, and Stx effects were evaluated in Vero cell cultures. The capacity to express active Stx1 and Stx2 toxins was determined in Vero cell cultures and the concentration of Stx was evaluated by 50% lethal dose (LD50) assays, observing inhibition of damaged cells by specific monoclonal antibodies. The results obtained in this study support the hypothesis that the stx gene is another putative virulence factor of Aeromonas, and since this gene can be transferred horizontally through OMVs this genus should be included as a possible causal agents of gastroenteritis and it should be reported as part of standard health surveillance procedures. Furthermore, these results indicate that the Aeromonas genus might be a potential causative agent of HUS.
ABSTRACT
INTRODUCTION: The increasing prevalence of uropathogenic Escherichia coli (UPEC) strains resistant to multiple antibiotics complicates the treatment of urinary tract infections (UTIs). This study aimed to analyze the antimicrobial resistance, serotypes, and phylogenetic groups among strains of E. coli isolated from outpatients with UTIs in Mexico City. METHODOLOGY: A total of 119 E. coli isolates were recovered from urine samples from outpatients with clinical diagnosis of uncomplicated UTIs from 2004 to 2007. The serotype was assessed by agglutination in microtiter plates; susceptibility to antimicrobials was determined by the disk diffusion method. Clone O25-ST131 and phylogenetic groups of E. coli strains were tested by methods based on PCR multiplex. RESULTS: The predominant serotype was O25:H4 (21.2%). Resistance to antibiotics was ampicillin (83.7%); piperacillin (53.8%); the fluoroquinolone group (55.5-60.6%), and trimethoprim/sulfamethoxazole (TMP/SMX) (56.4%). Additionally, 36 (30.2%) isolates were multidrug-resistant and 13 of these 36 strains were identified as E. coli O25-ST131 clone by an allele-specific PCR-based assay. Phylogenetic analysis showed that 15 of 17 isolates with serotype O25:H4 belonged to group B2. CONCLUSIONS: This is the first report that establishes the presence in Mexico of the O25-ST131 clonal group of E. coli, which has been associated with multidrug-resistance and with high virulence potential. The spread of this clone in Mexico should be monitored closely. We found a correlation between serotype O25:H4 and multidrug resistance in UPEC strains. Our results indicate that the use of ampicillin, fluoroquinolones, and TMP/SMX should be reviewed when selecting empirical therapy for UTIs.
Subject(s)
Drug Resistance, Bacterial , Escherichia coli Infections/epidemiology , Molecular Typing , Serotyping , Urinary Tract Infections/epidemiology , Uropathogenic Escherichia coli/classification , Uropathogenic Escherichia coli/isolation & purification , Adolescent , Adult , Aged , Agglutination Tests , Cluster Analysis , Escherichia coli Infections/microbiology , Female , Humans , Male , Mexico/epidemiology , Microbial Sensitivity Tests , Middle Aged , Molecular Epidemiology , Multiplex Polymerase Chain Reaction , Phylogeny , Urinary Tract Infections/microbiology , Young AdultABSTRACT
The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10(9) C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC(50) value of 200muM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/metabolism , Peptides/physiology , Peptidoglycan/biosynthesis , Uridine Diphosphate Sugars/metabolism , Amino Acid Sequence , Molecular Sequence Data , Pseudomonas aeruginosa/enzymologyABSTRACT
A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek®. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60 percent were identified as A. hydrophila, 20.4 percent as A. caviae, and 19.25 percent as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90 percent respectively) and environmental sources (45 and 10 percent respectively). Using "O" antisera, only 42.5 percent (94/221) of the strains were serologically identified, 55 percent (121/221) were non-typable, and 2.5 percent (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60 percent of the serotyped strains. More than 50 percent of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67 percent of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.