Afonso Gomes AbreuI; Sirlei Garcia MarquesII; Valério Monteiro-NetoIII, IV; Roberto Morais Luz de CarvalhoI; Azizedite Guedes GonçalvesI, IV
IPrograma de Pós-Graduação em Ciências da Saúde, Universidade Federal do Maranhão, São Luís, MA IIPrograma de Pós-Graduação em Ciências Biológicas (Microbiologia), Universidade Federal de Minas Gerais, Belo Horizonte, MG IIILaboratório de Pesquisa, Centro Universitário do Maranhão, São Luís, MA IVDepartamento de Patologia, Universidade Federal do Maranhão, São Luís, MA
ABSTRACT
INTRODUCTION: Extended spectrum β-lactamases (ESBLs) are enzymes that degrade β-lactam antibiotics and have been reported to be an important cause of nosocomial infection in worldwide.
METHODS: During 2009, 659 enterobacteria strains were isolated from different clinical specimens and tested for ESBL production. The disk approximation test, combined disk method and addition of clavulanic acid were used for phenotypic detection of the ESBL-producing strains and PCR for detection of the blaTEM and blaCTX-M genes.
RESULTS: Among the isolates, 125 were ESBL producers. The blaCTX-M and blaTEM genes were detected in 90.4% and 75% of the strains, respectively. Most strains were isolated from urine. Klebsiella pneumoniae was the most prevalent organism. Microorganisms presented high resistance to the antibiotics.
CONCLUSIONS: These results support the need for extending ESBL detection methods to different pathogens of the Enterobacteriaceae family because these methods are only currently standardized by the CLSI for Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis. Carbapenems were the antibiotic class of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae.
Keywords: Enterobacteriaceae. Antimicrobial resistance. ESBLs. Nosocomial infection.
RESUMO
INTRODUÇÃO: As β-lactamases de espectro ampliado (ESBLs) são enzimas que degradam os antibióticos β-lactâmicos e têm sido reportadas como uma importante causa de infecções hospitalares em todo o mundo.
MÉTODOS: Em 2009, 659 enterobactérias foram isoladas de diferentes espécimes clínicos e testadas quanto à produção de ESBL. Os testes de aproximação do disco, disco combinado e adição do ácido clavulânico foram utilizados na detecção fenotípica das amostras produtoras de ESBL e PCR para a detecção dos genes blaTEM e blaCTX-M.
RESULTADOS: Entre os isolados, 125 foram produtores de ESBL. Os genes blaCTX-M e blaTEMforam detectados em 90,4% e 75% das amostras, respectivamente. Com relação ao espécime clínico, a maioria das amostras foi isolada de urina. Klebsiella pneumoniae foi a espécie mais prevalente e o teste de susceptibilidade antimicrobiana mostrou uma elevada resistência dos microorganismos aos antibióticos testados.
CONCLUSÕES: Estes resultados suportam a necessidade de se ampliar os métodos de detecção das ESBLs para os diferentes patógenos da família Enterobacteriaceae, uma vez que esses métodos estão padronizados pelo CLSI apenas para Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca e Proteus mirabilis. Os carbapenens foram os antibióticos de escolha para o tratamento de infecções causadas por enterobactérias produtoras de ESBL.
Palavras-chaves: Enterobactérias. Resistência antimicrobiana. ESBLs. Infecção hospitalar.
INTRODUCTION
β-lactamases are a heterogeneous group of enzymes able to inactivate penicillins, cephalosporins and monobactams. These enzymes, frequently produced by aerobic and anaerobic Gram-negative bacteria, hydrolyze the ββ-lactam ring by irreversible hydroxylation, consequently inactivating the antibiotic1-3. Newly developed β-lactam antibiotics are specifically designed to be resistant to the hydrolytic action of β-lactamases. However, new β-lactamases have emerged because of the indiscriminate use of these antibiotics. A new class of antimicrobial agents, called oxyimino-cephalosporins or third-generation cephalosporins, were used as an alternative to treat severe infections caused by Gram-negative bacteria that produce restricted spectrum β-lactamases, such as TEM (Temoniera), TEM-1 and SHV-14.
The resistance mediated by β-lactamases emerged rapidly, and mutations in the blaTEM, blaTEM-1and blaSHV genes led to the emergence of novel enzymes, called extended-spectrum β-lactamases (ESBLs) due to their broad spectrum of activity, especially against oxyimino-cephalosporins5.
The production of ESBLs by enterobacteria is the most common resistance mechanism against β-lactams. These enzymes spread rapidly throughout the world and become the prevalent resistance mechanism once established in a region4,5. The prevalence of ESBL-producing strains in Latin American countries is 45%. Data from the SENTRY Antimicrobial Surveillance Program for Brazil show a high incidence of ESBL-producing isolates (Klebsiella pneumoniae: 50.3%, Escherichia coli: 9.1%)6. A high incidence of these strains is also observed in Europe (Klebsiella spp.: 32.8%, E. coli: 14.4%). A study conducted in Spain detected ESBL-producing strains in 90% of hospitals participating in a surveillance program. These findings demonstrate that the prevalence of ESBL-producing strains varies from country to country2,7,8.
Because of the increasing incidence of ESBL-producing Gram-negative bacteria and the lack of standardized phenotypic methods for the detection of ESBLs in a larger range of microorganisms, this study aimed to characterize ESBL-producing Enterobacteriaceae isolated at hospitals in northeast Brazil, focusing on the evaluation of their antimicrobial susceptibility profile.
METHODS
Bacterial strains
In two private hospitals and one public hospital in northeast Brazil, 659 enterobacteria strains collected from different clinical specimens between March and August 2009 were analyzed. All isolates were identified using conventional techniques9 and the Vitek 2 system, an automated assay (BioMérieux®, Marcy l’Etoile, France).
Antimicrobial susceptibility tests
Susceptibility testing was performed and interpreted via disk diffusion method, as recommended by the Clinical and Laboratory Standards Institute (CLSI)9, and the Vitek 2 system. Pseudomonas aeruginosa ATCC 27853, E. coli ATCC 25922 and ATCC 35218 were used as quality controls for antimicrobial susceptibility.
Detection of ESBL isolates
The disk approximation method and addition of clavulanic acid were employed for confirmation of ESBL phenotypes. Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively9.
Multiplex PCR amplification
PCR analysis was performed using sets of primers designed for amplification of genes codifying ESBLs. Primers TEM-164.SE (5′-ATGCGTTATATTCGCCTGTG-3′) and TEM-165.AS (5′-TGCTTTGTTATTCGGGCCAA-3′) were used for amplification of a 445-bp sequence of the TEM group. CTX-M-U1 (5′-ATGTGCAGYACCAGTAARGTKATGGC-3′) and CTX-M-U2 (5′-TGGGTRAARTARGTSACCAGAAYCAGCGG-3′) were used for amplification of a 593-bp sequence of the CTX-M group. Detection of the β-lactamase genes was carried out with the following components in a 25µl reaction volume: 1µl of DNA, 10pmol of the specific primers, 0.3µl of Taq DNA polymerase (0.5U/µl) (Invitrogen, Brazil), 1µl of MgCl2 (50mM) (Invitrogen, Brazil), 2.5µl of buffer (10x) (Invitrogen, Brazil), 2.5µl of dNTPs (2mM) (Invitrogen, Brazil) and Milli-Q water. PCR amplification conditions were as follows: initial denaturation step at 95°C for 15min, 30 cycles of denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 2min, followed by a final extension step at 72°C for 10min10.
Ethical considerations
Ethical approval was obtained from the Ethics Committee of the Universidade Federal do Maranhãoaccording to the requirements of the Ministry of Health.
RESULTS
Clinical bacterial strains
Among the 659 enterobacteria strains isolated from the 3 hospitals, 125 (19%) were determined to be as ESBL producers.
K. pneumoniae was the most frequent species (n = 63, 50.4%), followed by E. coli (n = 20, 16%). The largest number of ESBL-producing strains was isolated at hospital 2 (n = 64, 51.2%). In this hospital, Enterobacter aerogenes was the most frequent species (n = 14, 21.9%) when compared to hospitals 1 and 3. Table 1 shows the number of species isolated in each hospital.
Eighty-three (66%) of the isolates originated from ICUs, 27 (22%) from internal medicine units, 8 (6.4%) from surgical units, and 7 (5.6%) from outpatient clinics. In ICUs, K. pneumoniae was responsible for 56.6% of infections, followed by Proteus mirabilis (13.3%) and E. aerogenes(13.3%).
In the three hospitals, most ESBL-producing strains were isolated from urine (n = 45, 36%), followed by tracheal secretions (n = 25, 20%) and blood (n = 15, 12%).
Antimicrobial susceptibility of clinical isolates
Analysis of the antimicrobial susceptibility profile of the ESBL-producing strains showed that 100% of the isolates were resistant to ampicillin, ampicillin-sulbactam, cephalosporins and aztreonam. Most microorganisms had high resistance to ciprofloxacin, gentamicin, levofloxacin and trimethoprim-sulfamethoxazole but were susceptible to amikacin, piperacillin-tazobactam and carbapenems (ertapenem, imipenem and meropenem) (Table 2).
Characterization of ESBL
The presence of the two families of ESBL-coding genes was detected by PCR in 92% (115/125) of the isolates. The blaCTX-M gene was detected in 90.4% (104/115) of the isolates and the blaTEMgene in 75% (86/115). Seventy-five (65.2%) of the isolates carried genes encoding both the CTX-M and TEM-type enzymes (Table 3).
The highest frequency of genes encoding the CTX-M and TEM-type enzymes was observed in K. pneumoniae (96.6% [57/59] and 67.8% [40/59], respectively). The frequency of the blaCTX-Mgene was detected in 85% (17/20) of E. coli, 77.3% (11/15) of P. mirabilis, and 93.3% (14/15) of E. aerogenes isolates. The blaTEM gene was detected in 70.0% (14/20) for E. coli, 93.3% (14/15) for P. mirabilis, and 86.7% (13/15) for E. aerogenes (Table 4).
Although the susceptibility profile varied little between strains carrying the blaTEM and blaCTX-Mgenes, analysis showed that bacteria carrying the blaCTX-M gene were more susceptible to the antimicrobial agents tested (Table 5).
DISCUSSION
ESBL-producing bacteria have rapidly spread worldwide, indicating the need for continuous monitoring systems and effective control measures of infection. In addition, the therapeutic options for infections caused by ESBL-producing microorganisms are becoming increasingly more limited. The use of antibiotics, particularly oxyimino-cephalosporins, and hospital transfer are well-defined risk factors for the acquisition of ESBL-producing bacteria11.
The frequency of ESBL-producing Enterobacteriaceae detected was 19% (125/659), although rates of 29%12 and 24%13 have been reported in two other Brazilian studies. The rate of ESBL-producing microorganisms within the Enterobacteriaceae family is 11.1% in Poland14, 7.4% in Italy15, 6% in Saudi Arabia1, 1.7% in France16, and 30 to 60% in Latin America17. These data indicate that the prevalence of bacteria expressing the ESBL phenotype varies significantly in different regions and hospitals within the same region.
In this study, 37.5% (63/168) of the K. pneumoniae strains were producers of ESBLs, which supports similar results in other studies18. A frequency of approximately 50% is observed in Brazil, versus 5% in the United States and Japan, 15-20% in Europe, and 20-50% in Asian countries6,13,19,20.
Klebsiella pneumoniae was the most frequently observed ESBL-producing microorganism in this study. Similar results have been reported in members of the family Enterobacteriaceae7,13,18,19and in a multicenter study investigating bacterial resistance in Brazilian hospitals6. In contrast, E. coli was found to be the most prevalent ESBL-producing microorganism in Saudi Arabia1, France16and Italy15.
The occurrence of ESBL-producing E. aerogenes was similar to that of E. coli and P. mirabilis. In addition, the occurrence of this microorganism was higher than that of the other species in one of the hospitals (H2). The prevalence of E. aerogenes exceeded those found in Italy15. In contrast, other Brazilian studies have reported a higher prevalence of E. cloacae13.
Studies conducted in the United States indicate that standard ESBL testing in non-E. coli or non-Klebsiella spp. is not required because of the low phenotypic incidence21. However, our results demonstrated a high occurrence of non-E. coli, non-Klebsiella spp. and non-Proteus spp. Because of these findings and those of other studies3,13,21, standard detection techniques for ESBL enzymes in pathogens of the Enterobacteriaceae family are needed.
Our results agree with other studies that also found a high frequency of microorganisms isolated from urine1,22. However, in a study of various regions of Brazil, most ESBL-producing bacteria were isolated from blood and the respiratory tract6.
The main risk factors for colonization or infection with ESBL-producing bacteria are previous antibiotic use2,5, presence of invasive devices such as catheters23,24, prolonged hospital stay25,26, previous hospitalization17, delay in appropriate treatment, presence of ulcers5,23 and ICU stay2,17. The highest incidence of ESBL-producing bacteria was observed in ICUs, which can be explained by the emergent character of this unit. In addition, multiresistant bacteria dissemination occurs frequently because of the peculiar characteristics of this unit. These characteristics include the following: restricted unit, high frequency of healthcare worker contact with patients, increased possibility of pathogen cross-transmission, high selective pressure for broad-spectrum antibiotics, increased probability of environmental contamination (e.g., surgeries), use of medications that interfere with the natural chemical barrier or alter the immune response, and use of tubes and catheters that impair physiologic microorganism elimination27.
In general, the isolates presented high rates of antibiotic resistance, including resistance to other classes and cross-resistance. Some Brazilian studies have indicated fluoroquinolones as alternative drugs for the treatment of infections caused by ESBL-producing bacteria6. However, ESBL-producing bacteria were found to be highly resistant to these drugs in our results. Carbapenems were the most active drugs against ESBL-producing strains. These antibiotics can easily enter the bacteria and are more stable against hydrolysis mediated by ESBLs7. However, the administration of these drugs should be based on antimicrobial susceptibility testing. In the present study, two isolates (K. pneumoniae and E. cloacae) were found to be resistant to ertapenem and one (K. pneumoniae) was resistant to imipenem. These results were confirmed by the E-test method.
Most ESBLs evolved from gene mutations in classical ß-lactamases (TEM-1, TEM-2 and SHV-1), giving origin to ESBL varieties of mostly the TEM and SHV types5. A new family of ESBLs, CTX-M, has emerged over recent years, especially in E. coli. This family has become one of the most important families of ESBL enzymes in many countries28-30. CTX-M ß-lactamases are the predominant type of ESBLs in Europe and South America29,31, including Brazil32.
High frequencies of the blaCTX-M and blaTEM genes were discovered in this study. The frequency of the blaCTX-M gene was 90.4% (104/115). Other researchers also reported high prevalence rates of 92%33, 82%14, 72%34 and 70%10. The frequency of the blaTEM was 75% (86/115), which resembles the results of the studies conducted in Sweden and Brazil10,33.
A high occurrence of CTX-M-type ESBLs in E. coli has also been reported in recent studies16,30,34,35. However, the same was not observed for K. pneumoniae, as prevalence rates of 14.8% and 15.8% were reported in studies conducted in France16 and Norway35, respectively. The frequency of genes encoding CTX-M-type ESBLs is not restricted to E. coli. These genes are also observed in other species such as K. pneumonia, E. aerogenes and P. mirabilis.
Ten isolates (K. pneumoniae, n = 4; P. mirabilis, n = 3; Serratia marcescens, n = 2, and E. aerogenes, n = 1) identified as ESBL producers by the phenotypic methods did not produce the CTX-M or TEM enzyme. These strains probably produce other types of enzymes that were not investigated in this study.
In conclusion, the high percentage of ESBL-producing isolates detected in the three hospitals studied supports the need for extending the ESBL detection methods to different pathogens of the Enterobacteriaceae family. Currently, these methods are only standardized by the CLSI for E. coli, K. pneumoniae, K. oxytoca and P. mirabilis. CTX-M was the most prevalent enzyme in the ESBL-producing strains. Carbapenems remain the treatment of choice for infections caused by these pathogens.
ACKNOWLEDGMENTS
We express our gratitude to the Laboratório Cedro for supplying the isolates and to Dr. Libera Maria Dalla Costa from the Laboratório de Bacteriologia, Universidade Federal do Paraná, Brazil, for providing the TEM and CTX ESBL-producing E. coli controls.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
FINANCIAL SUPPORT
Brazilian Federal Agency for the Improvement of Higher Education (CAPES / Brazil).
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Address to:
Dra. Azizedite Guedes Gonçalves
Deptº Patologia/UFMA
Praça Madre Deus 02, Bairro Madre Deus
65025-560 São Luis, MA, Brasil.
Phone: 55 98 3235-0170; Fax: 55 98 3221-0270
e-mail: azizeg@ig.com.br
Received in 26/01/2011
Accepted in 28/02/2011
