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Introduction
Urinary tract infections (UTIs), both community-acquired and hospital-acquired, are among the most frequent infections in children and adults, thus representing a serious public health problem that results in a high economic burden for healthcare systems worldwide.1 UTIs are associated with high recurrence rates and when not adequately treated may progress rapidly to severe sepsis. These infections are mostly caused by bacteria of the Enterobacteriaceae family, primarily Escherichia coli. 2 , 3
Enterobacteriaceae pose a therapeutic challenge due to their rapid spread and their resistance to antibiotics.2,3 In recent years, there has been evidence of substantial changes in the susceptibility patterns of the most important bacterial microorganisms affecting the urinary tract, with a progressive increase in UTIs caused by extended-spectrum ß-lactamase (ESBL) -producing Enterobacteriaceae. 3,4 Unfortunately, the bacteria producing these enzymes are also resistant to other antimicrobials, which considerably reduces therapeutic options and has prompted further changes in the empirical antimicrobial treatment of these infections.4,5
Identifying these resistant phenotypes is a priority in clinical microbiology laboratories since such knowledge contributes to selecting the most appropriate antibiotic for treatment and helps to understand the extent of the problem and define strategies for controlling it.4,5 The epidemiological variation of UTIs is worrisome, as there is a worldwide increase in ESBL-producing bacteria that cause community-acquired urinary tract infections4,6with the morbidity, economic and social implications that this entails; Cuba is no exception to this problem.7,8
The prevalence of ESBLs varies depending on geographic location, with Latin America (44% for K. pneumoniae and 13.5% for E. coli) and Asia/Pacific (44% for K. pneumoniae and 12.0% for E. coli) being the regions with the highest prevalence, followed by Europe (13.3% for K. pneumoniae and 7.6% for E. coli) and North America (7.5% for K. pneumoniae and 2.2% for E. colt). 9 Furthermore, according to studies conducted in Peru, it can be established that the frequency of community-acquired ESBL-producing uropathogenic E. coli isolates increased from 16.30% in 201610 to 55.7% in 2021,11 which is significant.
Although data on this subject are scarce in Cuba, the results of certain studies point to a high prevalence of ESBL-producing E. coli as a cause of community-acquired UTIs. In 2014, Suárez-Trueba et al., 7 in a prospective descriptive study that included all E. coli strains isolated from urine cultures collected between March 1 and 31, 2012, at the Hospital Clínico Quirúrgico "Hermanos Ameijeiras" (n=131), established that ESBLs had a frequency of 30.3%. In turn, in 2015, Argüez de Paz et al., 8 in a descriptive observational study that included 150 urine cultures from patients treated between February 2011 and July 2013 in the urology service of the Hospital General Docente "Iván Portuondo" in San Antonio de Los Baños, found that 78.4% of the isolates were ESBLs.
In view of the foregoing, the objective of the present study was to determine the prevalence of ESBL-producing E. coli and K. pneumoniae isolates obtained from urine samples of outpatients with community-acquired UTIs in a Cuban municipality, as well as the antibiotic resistance profiles associated with the ESBL phenotype.
Materials and methods
Study design and type
Retrospective descriptive study conducted between January 1, 2019, and December 31, 2020.
Bacterial isolates
The study population comprised 2 004 urine cultures from adult patients (over 18 years of age) with a probable diagnosis of community-acquired UTI, which were obtained during the study period at the Clinical Microbiology Laboratory of the Hospital Clínico-Quirúrgico Docente Aleida Fernández Chardiet in the municipality of Güines. The final sample included only urine cultures in which E. coli and K. pneumoniae growths with counts >100 000 colony forming units (CFU)/mL were reported. On the other hand, urine cultures from pregnant women and cases in which growth of other uropathogens was found were excluded. Thus, the final sample consisted of 338 isolates (304 of E. coli and 34 of K. pneumoniae).
Procedures and instruments
Urine sample collection and isolate identification (genus and species) were performed following conventional methods as established in the standards and procedures recommended for microbiological diagnosis.12
Antimicrobial susceptibility testing
Antimicrobial susceptibility was determined using the Bauer-Kirby method following the Clinical Laboratory Standards Institute suggestions.13 The reading and interpretation of the zone of inhibition was also performed based on the criteria defined by this institution.
The antimicrobials considered in the study were: amoxicillin + clavulanic acid (AUG) 30/10µg, cefazolin (KZ) 30µg, cefoxitin (FOX) 30µg, cefotaxime (CTX) 30µg, ceftazidime (CAZ) 30µg, ceftriaxone (CRO) 30µg, cefepime (FEP) 30µg, aztreonam (ATM) 30µg, amikacin (AK) 30µg, ciprofloxacin (CIP) 5µg, and nitrofurantoin (F) 300pg.
Phenotypic detection of ESBL-producing isolates
Isolates considered as possible ESBL producers were those showing the following diameters in the zones of inhibition: ATM ≤27mm, CTX ≤27mm, CAZ ≤22mm, and CRO ≤25mm, which was confirmed through the double-disk diffusion test.
Double-disk diffusion test
The double-disk test was performed by placing the CTX, CAZ, CRO and ATM disks equidistant on the antibiogram at 2cm from an AUG disc. An enlarged or distorted zone of inhibition around one or all of the cephalosporin and ATM disks was interpreted as synergism. If such a deformation of the zone was present, the isolate was considered to be an ESBL-producing bacterium.13
The following control bacteria were used during the study: Escherichia coli ATCC 25922 as a negative control, and Klebsiella pneumoniae ATCC 700603 as a positive control.
Variables
Antimicrobial susceptibility testing interpretation categories
The isolates were classified into two interpretation categories: susceptible and resistant; the latter included isolates with intermediate susceptibility. Also, multidrug resistance was defined as resistance to antibiotics from 3 or more antibiotic families or groups.14
Statistical analysis
Data obtained from the isolates analyzed were entered into a Microsoft Excel database and described using absolute and relative frequencies. Furthermore, a bivariate analysis was performed using the chi-square test to determine differences in antibiotic resistance rates between ESBL-producing and non-producing bacteria, with a statistical significance level of p<0.05.
Ethical considerations
The study, which was approved by the Research and Ethics Committee of the Hospital Clínico-Quirúrgico Docente Aleida Fernández Chardiet according to Minutes No. 02/2021 of January 20, 2021, followed the ethical principles for research involving human subjects established in the Declaration of Helsinki.15 Likewise, anonymity of the participants' data was guaranteed and informed consent was not required because only isolates were analyzed.
Results
Phenotypic detection of ESBL
16.77% (51/304) and 17.64% (6/34) of the E. coli and K. pneumoniae isolates, respectively, were classified as ESBL-producing bacteria (Table 1).
Table 1 Frequency of Escherichia coli and Klebsiella pneumoniae isolates in extended-spectrum ß-lactamase-producing and non-producing enterobacteria.
| Isolates | Escherichia coli n (%) | Klebsiella pneumoniae n (%) |
|---|---|---|
| ESBL-producing bacteria | 51 (16.77%) | 6 (17.64%) |
| Bacteria that do not produce ESBL | 253 (83.23%) | 28 (82.36%) |
| Total | 304 (100%) | 34 (100%) |
ESBL: extended-spectrum ß-lactamase-producing enterobacteria.
Source: Own elaboration.
Antimicrobial resistance analysis
In general, resistance to the antibiotics considered was higher in ESBL-producing E. coli isolates than in non-producing ones, with statistically significant differences observed for all antibiotics with the exception of F (p>0.05). In the case of K. pneumoniae isolates, the frequency of antimicrobial resistance was higher for ESBL-producing isolates compared to non-producing isolates, with statistically significant differences observed in resistance to CTX, CRO, FEP, ATM, AK, and F (p <0.05). Detailed information is provided in Table 2.
Table 2 Distribution of antimicrobial resistance in Escherichia coli and Klebsiella pneumoniae isolates (extended spectrum ß-lactamase-producing and non-producing enterobacteria).
| Antibiotics | Escherichia coli no ESBL (n=253) n (%) | Escherichia coli ESBL (n=51) n (%) | p-value | Klebsiella pneumoniae no ESBL (n=28) n (%) | Klebsiella pneumoniae ESBL (n=6) n (%) | p-value |
|---|---|---|---|---|---|---|
| Amoxicillin + clavulanic acid | 175 (69.16) | 46 (90.19) | p<0.01 | 25 (89.28) | 5 (83.33) | p>0.05 |
| Cefazolin | 115 (45.45) | 50 (98.03) | p< 0.001 | 19 (67.85) | 6 (100.0) | p>0.05 |
| Cefotaxime | 63 (24.90) | 50 (98.03) | p< 0.001 | 16 (57.14) | 6 (100.0) | p<0.05 |
| Ceftazidime | 57 (22.52) | 50 (98.03) | p< 0.001 | 15 (53.57) | 5 (83.33) | p>0.05 |
| Ceftriaxone | 56 (22.13) | 51(100.0) | p< 0.001 | 15 (53.57) | 6 (100.0) | p<0.05 |
| Cefepime | 50 (19.76) | 51(100.0) | p< 0.001 | 13 (46.42) | 6 (100.0) | p<0.05 |
| Aztreonam | 61 (24.11) | 48 (94.11) | p< 0.001 | 15 (53.57) | 6 (100.0) | p<0.05 |
| Amikacin | 25 (9.88) | 22 (43.13) | p< 0.001 | 11 (39.28) | 3 (50.00) | p<0.05 |
| Ciprofloxacin | 98 (38.73) | 43 (84.31) | p< 0.001 | 17 (60.71) | 4 (66.66) | p>0.05 |
| Nitrofurantoin | 26 (10.27) | 7 (13.72) | p>0.05 | 10 (35.71) | 2 (33.33) | p<0.05 |
ESBL: extended-spectrum ß-lactamase-producing enterobacteria.
Source: Own elaboration.
Antimicrobial resistance profiles associated with the ESBL phenotype
According to the distribution of resistance profiles, ESBL-producing E. coli isolates (n=51) were grouped into 4 resistance patterns (Table 3), with pattern III (ESBL+CIP) being the most frequent (43.13%), followed by pattern II (ESBL+CIP, AK) (27.45%). In addition, 41.17% of the isolates were multidrug-resistant.
Table 3 Antimicrobial resistance profile in Escherichia coli isolates producing extended-spectrum ß-lactamase-producing enterobacteria.
| Resistance pattern | Resistance profiles | n (%) |
|---|---|---|
| I | ESBL + CIP, AK, F | 7 (13.72%) |
| II | ESBL + CIP, AK | 14 (27.45%) |
| III | ESBL + CIP | 22 (43.13%) |
| IV | ESBL + AK | 1 (1.96%) |
| Multi-resistant isolates | 21 (41.17%) |
ESBL: extended spectrum ß-lactamase-producing enterobacteria; AK: amikacin; CIP: ciprofloxacin; F: nitrofurantoin.
Source: Own elaboration.
In ESBL-producing K. pneumoniae isolates (n=6), 3 resistance patterns were identified, with a predominance of pattern I (ESBL + CIP, AK, F) (33.33%). There were 3 multi-drug-resistant isolates (50.00%) (Table 4).
Table 4 Antimicrobial resistance profile in Klebsiella pneumoniae isolates producing extended-spectrum ß-lactamase-producing enterobacteria.
| Resistance pattern | Resistance profiles | n (%) |
|---|---|---|
| I | ESBL + CIP, AK, F | 2 (33.33%) |
| II | ESBL + CIP, AK | 1 (16.66%) |
| III | ESBL + CIP | 1 (16.66%) |
| Multi-resistant isolates | 3 (50.00%) |
ESBL: extended spectrum ß-lactamase-producing enterobacteria; AK: amikacin; CIP: ciprofloxacin; F: nitrofurantoin.
Source: Own elaboration.
Discussion
ESBLs are a mechanism of antibiotic resistance in bacteria of the Enterobacteriaceae family2,3 and, in recent years, an increasing number of reports have demonstrated their presence in isolates of community-acquired infections.4-11
In the present study, the prevalence of ESBL-producing E. coli was 16.77%, a figure higher than that reported in studies of ESBL detection in uropathogenic E. coli isolates from Brazil (7.1%),16 Colombia (12.52%),17 and Paraguay (13%).18 Nevertheless, the prevalence described in the present study is similar to the 16.30% reported in 2012 by Galván et al. 10 in a phenotypic and molecular characterization study that included 53 E. coli isolates from outpatients in Lima, Peru, and to the 17.92% described in 2016 by Alviz-Amador et al. 19 in a cross-sectional study that included 396 uropathogen-positive urine cultures, in which the susceptibility profile of a hospital in Cartagena, Colombia, was determined. However, in a study conducted by Miranda et al. 20 on 1 026 E. coli isolates from adult patients treated at a private clinic in Lima between January 2014 and October 2016 (927 outpatients and 99 inpatients), a higher prevalence than that of the present study was reported: 41.7% overall, 40.8% for outpatients, and 50.5% for inpatients.
In the Cuban case, Suárez-Trueba et al., 7 in a study on antibiotic susceptibility and resistance mechanisms carried out on 131 isolates of E. coli obtained from urine cultures of patients treated at a tertiary hospital in Havana, reported that the proportion of ESBL-producing phenotypes in isolates from outpatients (n=11) was 30.3%. However, Argüez de Paz et al., 8 in a study on beta-lactamase production carried out in San Antonio de Los Baños that included 150 positive urine cultures (51 of E. coli and 22 of K. pneumoniae) from patients with UTIs, reported that the prevalence of ESBL among E. coli isolates was 78.4.
With regard to K. pneumoniae isolates, in the present study the ESBL phenotype was detected in 17.6% of the isolates, a figure higher than the 3.54% reported in 2016 by Alviz-Amador et al. 19 in Colombia, but much lower than the 77.2% described by Argüez de Paz et al. 8 This finding was also much lower than the one reported by Miranda et al., 20 whose study included 108 K. pneumoniae isolates (93 outpatients and 15 inpatients), with an overall prevalence of these microorganisms of 50.92% (53.8% for outpatients and 33.3% for inpatients).
As mentioned above, the prevalence of ESBL-producing E. coli and K. pneumoniae varies from country to country, and even among studies, as is the case between the present study and others conducted in Cuba.7,8This phenomenon could be explained by differences in the sample sizes of the studies, the methods used for detection, the previous use of antimicrobials in the included patients, and the patients' own risk factors. These variations have serious therapeutic implications, as they make it difficult to establish a standard treatment for UTIs since these bacteria present intrinsic resistance to beta-lactams and sometimes co-resistance with other first-line antibiotics (fluoroquinolones and aminoglycosides). Therefore, it is suggested to be aware of the importance of an appropriate reading of the antibiogram to identify ESBL-producing bacteria at an early stage and, consequently, to implement an adequate use of antibiotics and take pertinent measures to control the development of infections by this type of microorganisms, both in the community and in the hospital environment.
Furthermore, in the present study, 84.31% of ESBL-producing E. coli isolates were found to be resistant to IPC, with a significant difference with non-ESBL-producing E. coli isolates (p<0.001). This figure was much higher than the 26.5% resistance to this antibiotic found in 28 ESBL-producing E. coli isolates by Marcos-Carbajal et al., 21 who conducted a comparative study evaluating the antimicrobial resistance profiles of uropathogenic E. coli and the incidence of ESBL production in 98 E. coli isolates collected in 3 private health care facilities in Peru, or the 40% reported by Montañez-Valverde et al. 22 in a study conducted in Peru with 81 ESBL-producing E. coli isolates from patients with UTI, but the figure is lower than the 97.3% described by Alviz-Amador et al. 19 in Colombia in isolates with this phenotype. Likewise, in the present study, 43.13% of ESBL-producing E. coli isolates were resistant to AK, a result that differs from the low levels of resistance to this antibiotic reported by Montañez-Valverde et al. 22 and Alviz-Amador et al., 19 which were 3.5% and 12%, respectively. The high resistance rates to IPC and AK have a clinical impact, limiting their use in patients with infections caused by ESBL-producing bacteria.
Likewise, in the present study, 66.66% of ESBL-producing K. pneumoniae isolates showed resistance to CIP. This finding is similar, although slightly lower, to the 77.78% resistance rate to this antibiotic found in 27 ESBL-producing K. pneumoniae isolates analyzed in the study by Chero-Vargas et al., 23 which was conducted using data from 567 urine cultures from older adults (60 years or older) treated in 2017 at a private clinic in Lima in different levels of care (emergency, outpatient, inpatient, and ICU).
Similarly, in the present study, AK resistance was observed in 50% of ESBL-producing K. pneumoniae isolates. This is much higher than the 18.5% of resistance to this antibiotic in this type of isolates reported by Chero-Vargas et al., 23 but similar to the 43.3% found in 12 ESBL-producing K. pneumoniae isolates described by Guevara et al., 24 in a study on antimicrobial susceptibility patterns of gram-negative bacteria isolated from urinary tract infections in Venezuela.
Finally, the results concerning resistance profiles in ESBL-producing E. coli and K. pneumoniae isolates reported here, i.e., the change in the resistance profile, can be explained by many reasons, including indiscriminate use of antibiotics, spontaneous mutations, transfer of deoxyribonucleic acid (DNA) between bacteria, among others. This finding may indicate the possible circulation of genetic elements that encode information about resistance mechanisms to several families of antimicrobials, an aspect that should be corroborated in further studies.
Notably, the main limitation of the present study was that virulence and molecular studies (determination of genes encoding ESBLs) were not performed.
Conclusions
The results of the present study, the first of its kind to be carried out in Güines, confirm the presence of ESBL-producing E. coli and K. pneumoniae in uropathogenic isolates obtained from patients with community-acquired UTIs in this municipality. These results should be considered as a warning to health authorities about the need to establish the real prevalence in other regions of the country in order to implement the necessary actions to control the circulation of multidrug-resistant bacteria, carry out studies on risk factors in outpatients, and develop policies to prevent infections caused by this type of bacteria.
