Artículo original

 

Risk factors for extended-spectrum β-lactamases-producing Escherichia coli urinary tract infections in a tertiary hospital

 

Factores de riesgo en infecciones de vías urinarias causadas por Escherichia coli productora de β-lactamasas de espectro extendido en un hospital de tercer nivel

 

María Dolores Alcántar-Curiel, DSc,⁽¹⁾ Celia Mercedes Alpuche-Aranda, MD, DSc,⁽²⁾ Héctor Javier Varona-Bobadilla, MD,⁽³⁾ Catalina Gayosso-Vázquez, BSc,⁽¹⁾ Ma. Dolores Jarillo-Quijada, BSc,⁽¹⁾ Mauricio Frías-Mendivil, MD,⁽⁴⁾ Lucio Sanjuan-Padrón, MD,⁽³⁾ José Ignacio Santos-Preciado, MD, DSc.⁽¹⁾

 

(1) Facultad de Medicina, Universidad Nacional Autónoma de México. Distrito Federal, México.

(2) Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Salud Pública. Cuernavaca, Morelos, México.

(3) Hospital General Naval de Alta Especialidad, Secretaría de Marina Armada de México. Distrito Federal, México.

(4) Universidad Estatal de Sonora. Hermosillo, Sonora, México.

 

Corresponding author

 

---

Abstract

Objective. To assess the risks factors for urinary tract infections (UTIs) caused by Extended-Spectrum Beta-Lactamases (ESBLs)-producing E. coli and the molecular characterization of ESBLs.

Materials and methods. A case-control study was performed to identify risk factors in consecutively recruited patients with UTIs caused by ESBLs or non-ESBLs-producing E. coli in a tertiary hospital in Mexico.

Results. ESBLs-producing E. coli were isolated from 22/70 (31%) patients with E. coli UTIs over a three month period. All isolates were resistant to cephalosporins and quinolones but susceptible to carbapenems, amikacin and nitrofurantoin. Prior antibiotic treatment with more than two antibiotic families (OR=6.86; 95%CI 1.06-157.70; p=0.028), recurrent symptomatic UTIs (OR=5.60; 95%CI 1.88-17.87; p=0.001) and previous hospitalization (OR=5.06; 95%CI 1.64-17.69; p=0.002) were significant risk factors. Sixteen isolates harbored the beta-lactamase (bla)_CTX-M-15 gene and five the bla_TEM-1 gene.

Conclusions. One of every three patients presented UTIs with ESBLs-producing beta-lactams and fluoroquinolone resistant E. coli. Risk factors and resistance patterns must be taken into account for developing antibiotic use policies in these settings.

Key words: Escherichia coli; beta-lactamases; urinary tract infections; risk factors; Mexico.

---

Resumen

Objetivo. Evaluar los factores de riesgo en infecciones de vías urinarias (IVUs) causadas por E. coli productora de Beta-Lactamasas de espectro extendido (BLEEs) y caracterizar las BLEEs.

Material y métodos. Estudio de casos y controles en pacientes consecutivos con IVUs causadas por E. coli productoras o no de BLEEs en un hospital de referencia.

Resultados. E. coli productora de BLEEs se aisló en 22/70 (31%) pacientes con IVUs por E. coli durante un periodo de tres meses. Todos los aislamientos fueron resistentes a cefalosporinas y quinolonas, pero susceptibles a carbapenemes, amikacina y nitrofurantoina. Factores de riesgo significativos incluyeron tratamiento previo con más de dos familias de antibióticos (OR=6.86; IC95% 1.06-157.70; p=0.028), IVUs sintomáticas recurrentes (OR=5.60; IC95% 1.88-17.87; p=0.001) y hospitalizaciones previas (OR=5.06; IC95% 1.64-17.69; p=0.002). Dieciséis aislamientos presentaron el gen betalactamasas (bla)_CTX-M-15 y cinco el gen bla_TEM-1.

Conclusiones. Uno de cada tres pacientes presentó IVU con E. coli resistente a beta-lactámicos, fluoroquinolonas y productora de BLEEs. En estos casos, los factores de riesgo y patrones de resistencia deberían tomarse en cuenta para recomendar tratamiento.

Palabras clave: Escherichia coli; beta-lactamasas; infecciones de vías urinarias; factores de riesgo; México.

---

 

Urinary tract infections (UTIs) are an important cause of morbidity in the general population, being Escherichia coli the principal etiologic agent.¹ At present the production of extended spectrum beta-lactamases (ESBLs) by this uropathogen complicates treatment because their presence not only implies resistance to beta-lactam antibiotics but is also associated with resistance to other families of antibiotics.²

ESBLs production is one of the main contributors to the problem of antimicrobial resistance. ESBLs are enzymes produced by gram-negative bacilli, most commonly derived from TEM or SHV parents, but the prevalence of CTX-M types has increased dramatically since 1998 in most parts of the world.³ Typically, the isolation of ESBLs-producing E. coli has occurred in the hospital setting, but this organism has begun to disseminate in the community.⁴

Numerous studies have investigated the risk factors for UTIs caused by ESBLs-producing E. coli in the world, however, they are limited in Mexico.^5-7 The identification of predisposing factors is relevant to the prevention of this common infectious disease diagnosed in outpatients. The increasing prevalence of ESBLs-producing E. coli prompted our interest to investigate risk factors for ESBLs-producing E. coli in patients with community-onset UTIs. The aims of this study were to identify potential risk factors for hospital or community acquired UTIs by ESBLs-producing E. coli, determine the prevalence of antimicrobial resistance, identification and molecular characterization of genes encoding ESBLs and to determine the clonal relationship between clinical isolates.

 

Materials and methods

Study design and population

A case-control study was conducted to identify risk factors in consecutively recruited patients who presented with UTI caused by ESBLs-producing E. coli to Hospital General Naval de Alta Especialidad, Secretaría de Marina Armada de México (Hosgenaes), Mexico's Naval Referral Hospital, a 150 bed tertiary care teaching hospital, which provides medical services to the naval branch of Mexico's armed forces. The study was approved by the hospital's research and ethics commission. The diagnosis of UTIs was defined by the presence of symptom related to the urinary tract, pyuria (≥10 leucocytes per high-power field) and positive clean catched urine culture [≥10⁵ colony-forming units (CFU)/mL] of E. coli.⁸ Only the first isolate from each patient was included in the analysis. Adult patients with UTIs due to ESBLs-producing E. coli (cases) and non-ESBLs-producing E. coli (controls) were prospectively identified through records from the hospital's clinical microbiology laboratory over a three month period, August to October, 2011. Controls were matched in a 2:1 ratio to case patients and potential risk factors were recorded by abstracting medical records. A case was identified as an inpatient or outpatient with symptoms related to UTIs and a positive urine culture for ESBLs-producing E. coli. Controls were patients with UTIs due to non-ESBLs-producing E. coli. Clinical information included age, gender, previous hospitalization (within the past year), recent antibiotic treatment within 60 days, number of antibiotic treatments, antimicrobial regimen for UTIs, recurrent symptomatic urinary tract infections, underlying diseases (urological abnormalities, diabetes mellitus), and use of indwelling urinary catheter.

Bacteria, antibiotic susceptibility, and ESBLs production

Identification, susceptibility pattern, and ESBLs production were initially detected by the Phoenix system (Becton Dickinson, USA). Susceptibility profile of the isolates against to 22 antibiotics was determined using a disk diffusion method as the CLSI guidelines⁹ and determining minimum inhibitory concentration (MIC) by BIOMIC System (Giles Scientific, USA). The ESBLs-producer E. coli was confirmed for ceftazidime and cefotaxime with or without clavulanate by double disk synergism method.⁹ Klebsiella pneumoniae ATCC 700603 was used as control in all assays. An increase of >5 mm in a zone diameter for either antimicrobial agent tested in combination with clavulanate and their respective zone diameter of the agents when tested alone confirmed the presence of ESBLs-producing isolates.⁹

Detection and characterization of bla genes

PCR was used to detect the bla_CTXM, bla_SHV, and bla_TEM genes using previously described primers.^10-12 Amplified products were subjected to nucleotide sequencing at the Instituto de Biotecnología, Universidad Nacional Autónoma de México and analyzed using Clone Manager suite 7.0 and Basic Local Alignment Search Tool (BLAST).

Pulsed Field Gel Electrophoresis

A possible clonal relationship among the isolates was determined. Genotyping of ESBLs-producing E. coli was performed by Pulsed Field Gel Electrophoresis (PFGE), as described previously in a standard protocol established by the Centers for Disease Control and Prevention (PulseNet; Centers for Disease Control and Prevention 2008), using genomic DNA and enzymatic restriction analysis with XbaI. The DNA band patterns were analyzed using Tenover criteria.¹³ The percentage of similar profiles was calculated using the Dice coefficient,¹⁴ isolates with a Dice coefficient correlation >85% were considered to belong to the same pulsotype (clone).

Statistical analysis

Qualitative variables were compared using chi square test. Logistic regression models were used to calculate odds ratios and 95% confidence intervals (CI). For all comparison, a p value of <0.05 was considered statistically significant.

 

Results

A total of 70 patients with UTIs caused by E. coli were recruited during the study period. Confirmatory double disc synergy diffusion test showed that 22 (31%) isolates were phenotypic positive for ESBLs-production (one in three patients with UTIs), 19 of these isolates came from the outpatient services and the other three isolates from inpatient (Surgery, Internal Medicine and Intensive Care Unit).

Univariate and multivariate analysis were performed with patient characteristics, epidemiological data and variables associated with development of UTIs caused by ESBLs-producing E. coli (table I). Gender and mean age were not found to be statistically significant risk factors between the case and control groups. Previous exposure to antibiotics (OR=6.86; 95%CI 1.06-157.70; p=0.028), recurrent symptomatic UTIs (OR=5.60; 95%CI 1.88-17.87; p=0.001), use of >2 of different families of antibiotics (OR, 5.14; 95%CI 1.31-11.47; p=0.0), previous hospitalization (OR=5.06; 95%CI 1.64-17.69; p=0.002), urological abnormalities (OR=3.88; 95%CI 1.31-11.47; p=0.005), and urinary catheterization (OR=3.90; 95%CI 1.13-14.08; p=0.008), were significantly associated with development of ESBL-producing E. coli UTIs.

Susceptibility phenotype of ESBLs-producing E. coli isolates are summarized in table II. As expected, multidrug-resistant comprised 100% of these isolates, they were resistant to three or more different families of antibiotics; aminopenicillins, third-generation cephalosporins and fluoroquinolones. Nevertheless, all isolates remained fully susceptible to carbapenems and highly susceptible to amikacin and nitrofurantoin.

PCR amplification of DNA from 22 ESBL-producing E. coli isolates showed the presence of two ESBLs families; CTX-M in 16/23 (70%) isolates and TEM in 5/23 (22%) isolates (four isolates from outpatients and one from inpatient), SHV was not detected. Sequencing of CTX PCR products demonstrated 100% homology to the sequence CTX-M-15, GenBank accession no. AY044435, and TEM PCR products demonstrated 100% homology to the sequence TEM-1 GenBank accession no. J01749. One isolate from the Surgery ward and one isolate from an outpatient simultaneously harbored bla_CTX-M-15 and bla_TEM-1 genes. Four isolates with ESBLs production were negative for bla genes studied.

PFGE analysis of 22 ESBLs-producing E. coli isolates yielded 14 different clones and one isolate was not determined. These results indicate a high clonal variability (figure 1), and close genetic relatedness among five of the isolates belonging to clone E. These five isolates were from outpatients, three of them with a history of previous hospitalization. Additionally, these five isolates presented identical resistance patterns to 16/21 antimicrobials tested (included Beta-lactams, Beta-lactams-Beta-lactamase inhibitors combinations, aminoglycosides, and fluoroquinolones) and carried only the bla_CTX-M-15.

 

Discussion

E. coli remains as one of the most important ESBLs-producing bacteria in human infections worldwide. Several studies have addressed the impact of risk factors associated with community-onset UTIs caused by ESBLs-producing E. coli.^15-17 The most common and significant risks factors associated with these infections included recent hospitalization,^15-17 prior use of cephalosporins,^16,17 catheterization,^15,16,18 and diabetes.^19,20 However, studies on risk factors for infections due to ESBLs-producing E. coli in Mexico are limited and have been related to bloodstream and surgical wound infections.^5-7,21 Our results are similar to reports in other parts of the world and show that the previous use and the number of different families of antibiotics used, mainly cephalosporins, along with prior hospital stay, are major risk factors for acquisition of the UTIs caused by ESBLs-producing E. coli.^15-20,22

In this prospective case-control study, ESBLs-producing E. coli was isolated in 33% of patients with UTI (78% of these cases were outpatients), a prevalence comparable to that found in a previous report from Latin American countries.²³ These isolates were 100% resistant to aminopenicillins, cephalosporins and fluoroquinolones, thereby the management of related UTIs could be more difficult. However, results in our study showed that amikacin, carbapenem and nitrofurantoin have in vitro efficacy. Because of the risk of nephrotoxicity and ototoxicity in the elderly,²⁴ amikacin should be used in short term therapy²⁴ and for that reason nitrofurantoin and carbapenems may be the antibiotics of choice in the treatment of these infections.

The types of ESBLs present in isolates in this hospital were CTX-M-15 and TEM-1 in 70% and 22% of the isolates, respectively. These results correlate with ESBLs families most commonly associated with ESBL-producing E. coli causing community-onset UTIs in most areas of the world.^5,7,17.25-30 Because four strains were phenotypically negative to the bla_CTX-M, bla_TEM and bla_SHV genes, these results suggest the involvement of other ESBLs genes that were not tested for in this study. For example, bla_VEB gen has been reported within the genes identified from ESBL-producing E. coli isolated from community-onset UTIs in Taiwan³¹ and bla_TLA-1 gen has been identified in 11% of the ESBL-producing Enterobacteriaceae causing nosocomial infections in Mexico.³²

Although clonal variation was observed in the isolates studied, clone E was the most frequent, including five isolates from the community, three of them from patients with a recent history of previous hospitalization, which lead us to hypothesize that these isolates were acquired previously in the hospital.

The molecular analysis of the isolates in this study, which revealed the presence of CTX-M-15 and TEM-1 in different clones, suggests a likely horizontal dissemination of plasmids between ESBLs-producing E. coli and the association with multidrug-resistance observed. This is the first report to document the presence of CTX-M-15 and TEM-1-producing multi-drug-resistant E. coli causing UTIs in Mexico.

What are the clinical implications of these finding? When ESBLs-producing isolates are resistant to second- and third-generation cephalosporins, the treatment options are limited to carbapenems, aminoglycosides and nitrofurantoin.¹⁶ Our findings strongly suggest that in this hospital setting, carbapenems and nitrofurantoin could be selected as empirical treatment options for out- and inpatients with ESBLs-producing E. coli UTIs and as these patients are at risk of developing sepsis, carbapenems but not nitrofurantoin could also be considered in the empirical treatment of bloodstream infections in patients with underlying³³ or recent history of E. coli urinary tract infection.

 

Acknowledgments

This work was supported in part by grant No. 2003-C1-63 from the Consejo Nacional de Ciencia y Tecnología (Conacyt), México.

 

References

1. Kaper JB, Nataro JP, Mobley HT. Pathogenic Escherichia coli. Nature Rev 2004;2:124-40.         [ Links ]

2. Dhillon, R, Clark J. ESBLs: a clear and present danger. Crit Care Res Pract 2012; 2012:625170. doi: 10.1155/2012/625170.         [ Links ]

3. Paterson DL, Bonomo RA. Extended-spectrum ß-lactamases: a clinical update. Clin Microbiol Rev 2005;18:657-686.         [ Links ]

4. Datta P, Gupta V, Sidhu S. Extended spectrum beta lactamase positive uropathogenic E. coli epidemiological factors and resistance. BJMP 2014;7:a718.         [ Links ]

5. Cornejo-Juárez P, Pérez-Jiménez C, Silva-Sánchez J, Velázquez-Acosta C, González-Lara F, Reyna-Flores F, et al. Molecular analysis and risk factors for Escherichia coli producing extended-spectrum ß-lactamase bloodstream infection in hematological malignances. PLOS One 2012;7:e35780.         [ Links ]

6. Montes CV, Vilar-Compte D, Velázquez C, Golzarri MF, Cornejo-Juárez P, Larson EL. Risk factors for extended spectrum ß-Lactamase-producing Escherichia coli versus susceptible E. coli in surgical site infections among cancer patients in Mexico. Surg Infect 2014;15:627-634.         [ Links ]

7. Muro S, Garza-González E, Camacho-Ortiz A, González GM, Llaca-Díaz JM, Bosques F, Rositas F. Risk factors associated with extended-spectrum ß-lactamase-producing Enterobacteriacea nosocomial bloodstream infections in a tertiary care hospital: a clinical and molecular analysis. Chemotherapy 2012;58:217-224.         [ Links ]

8. Bouza E, San Juan R, Munoz P, Voss A, Kluytmans J. Cooperative Group of the European Study Group on Nosocomial Infections. European perspective on nosocomial urinary tract infections II. Clin Microbiol Infect 2001;7(10):532-542.         [ Links ]

9. CLSI Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI Document M100-S24. Wayne, PA: Clinical and Laboratory Standards Institute, 2014.         [ Links ]

10. Rasheed JK, Jay C, Metchock B, Berkowitz F, Weigel L, Crellin J, et al. Evolution of extended-spectrum ß-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia. Antimicrob Agents Chemother 1997;41:647-653.         [ Links ]

11. Siu LK, Lu P, Hsueh P, Lin FM, Chang S, Luh K, et al. Bacteremia due to extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in a pediatric oncology ward: clinical features and identification of different plasmids carrying both SHV-5 and TEM-1 genes. J Clin Microbiol 1999;37:4020-4027.         [ Links ]

12. Dutuor C, Bonnet R, Marchandin H, Boyer M, Chanal C, Sirot D, et al. CTX-M-1, CTX-M-3, and CTX-M-14 ß-Lactamases from Enterobacteriaceae isolated in France. Antimicrob Agents Chemother 2002:46:534-537.         [ Links ]

13. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-2239.         [ Links ]

14. Dice LR. Measures of the amount of ecologic association between species. Ecology 1945;26:299e302.         [ Links ]

15. Wu YH, Chen PL, Hung YP, Ko WC. Risk factors and clinical impact of levofloxacin or cefazolin nonsusceptibility or ESBL production among uropathogens in adults with community-onset urinary tract infections. J Microbiol Immunol Infect 2014;47:197-203.         [ Links ]

16. Kizilca O, Siraneci R, Yilmaz A, Hatipoglu N, Ozturk E, Kiyak A, et al. Risk factors for community-acquired urinary tract infection caused by ESBL-producing bacteria in children. Pediatr Int 2012;54:858-862.         [ Links ]

17. Calbo E, Romaní V, Xercavins M, Gómez L, Vidal CG, Quintana S, et al. Risk factors for community-onset urinary tract infections due to Escherichia coli harbouring extended-spectrum beta-lactamases. J Antimicrob Chemother 2006;57:780-783.         [ Links ]

18. Al-Assil B, Mahfoud M, Hamzeh AR. Resistance trends and risk factors of extended spectrum ß-lactamases in Escherichia coli infections in Aleppo, Syria. Am J Infect Control 2013;41:597-600.         [ Links ]

19. Inns T, Millership S, Teare L, Rice W, Reacher M. Service evaluation of selected risk factors for extended-spectrum beta-lactamase Escherichia coli urinary tract infections: a case-control study. J Hosp Infect 2014;88:116-119.         [ Links ]

20. Briongos-Figuero LS, Gómez-Traveso T, Bachiller-Luque P, González MD, Gómez-Nieto A, Palacios-Martín T, et al. Epidemiology, risk factors and comorbidity for urinary tract infections caused by extended-spectrum beta-lactamase (ESBL)-producing enterobacteria. Int J Clin Pract 2012;66:891-896.         [ Links ]

21. Mosqueda-Gómez JL, Montaño-Loza A, Rolón AL, Cervantes C, Bobadilla-del-Valle JM, Silva-Sánchez J, et al. Molecular epidemiology and risk factors of bloodstream infections caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae. A case-control study. Inter J Infect Dis 2008;12:653-659.         [ Links ]

22. Quirante OF, Cerrato SG, Pardos SL. Risk factors for bloodstream infections caused by extended-spectrum betalactamase-producing Escherichia coli and Klebsiella pneumoniae. Braz J Infect Dis 2011;15:370-376.         [ Links ]

23. Bours PHA, Polak R, Hoepelman AIM, Delgado E, Jarquin A, Matute AJ. Increasing resistance in community-acquired urinary tract infections in Latin America, five years after the implementation of national therapeutic guidelines. Inter J Infect Dis 2010;14:e770-e774.         [ Links ]

24. Khawcharoenporn T, Vasoo S, Singh K. Urinary tract infections due to multidrug-resistant enterobacteriaceae: prevalence and risk factors in a Chicago emergency department. J Emerg Med 2013;2013:258517.         [ Links ]

25. Oteo J, Pérez-Vázquez M, Campos J. Extended-spectrum ß-lactamase producing Escherichia coli: changing epidemiology and clinical impact. Curr Opin Infect Dis 2010;23:320-326.         [ Links ]

26. Freeman JT, Rubin J, McAuliffel GN, Peirano G, Roberts SA, Drinković D, et al. Differences in risk-factor profiles between patients with ESBL-producing Escherichia coli and Klebsiella pneumoniae: a multicentre case-case comparison study. Antimicrob Resist Infec Control 2014;3:27.         [ Links ]

27. Hayakawa K, Gattu S, Marchaim D, Bhargava A, Palla M, Alshabani K, et al. Epidemiology and risk factors for isolation of Escherichia coli producing CTX-M-type extended-spectrum ß-lactamase in a large U.S. medical center. Antimicrob Agents Chemother 2013;57:4010-4018.         [ Links ]

28. Leistner R, Meyer E, Gastmeier P, Pfeifer Y, Eller C, Dem P, et al. Risk factors associated with the community-acquired colonization of extended-spectrum beta-lactamase (ESBL) positive Escherichia coli. An exploratory case- control study. PLOS ONE 2013;8:e74323.         [ Links ]

29. Søraas A, Sundsfjord A, Sandven I, Brunborg C, Jenum PA. Risk factors for community-acquired urinary tract infections caused by ESBL-producing Enterobacteriaceae -A case-control study in a low prevalence country. PLoS One 2013;8:e69581.         [ Links ]

30. Yaita K, Aoki K, Suzuki T, Nakaharai K, Yoshimura Y, Harada S, et al. Epidemiology of extended-spectrum ß-lactamase producing Escherichia coli in the stools of returning japanese travelers, and the risk factors for colonization. PLOS ONE 2014;9:e98000.         [ Links ]

31. Kung CH, Ku WW, Lee CH, Fung CP, Kuo SC, Chen TL, Lee YT. Epidemiology and risk factors of community-onset urinary tract infection caused by extended-spectrum ß-lactamase-producing Enterobacteriaceae in a medical center in Taiwan: a prospective cohort study. J Microbiol Immunol Infect. 2015;48(2):168-174.         [ Links ]

32. Silva-Sánchez J, Garza-Ramos JU, Reyna-Flores F, Sánchez-Pérez A, Rojas-Moreno T, Andrade-Almaraz V, et al. Extended-spectrum ß-lactamase-producing Enterobacteriacea causing nosocomial infections in Mexico. A retrospective and multicenter study. Arch Med Res 2011;42:156-162.         [ Links ]

33. Pitout JDD, Laupland KB. Extended-spectrum ß-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159-166.         [ Links ]

 

Received on: March 23, 2015
Accepted on: June 26, 2015

 

Corresponding author:
José Ignacio Santos-Preciado.
Unidad de Investigación en Medicina Experimental,
Facultad de Medicina, Universidad Nacional Autónoma de México.
Dr. Balmis 148, Col. Doctores. 06720 Distrito Federal, México.
E-mail: joseignaciosantos56@gmail.com

Declaration of conflict of interest. The authors declare that they have no conflict of interests.