Nosocomial infection and characterization of extended-spectrum β-lactamases-producing Enterobacteriaceae in Northeast Brazil

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.

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 β-lactam ring by irreversible hydroxylation, consequently inactivating the antibiotic [1][2][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 oxyiminocephalosporins 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-1 4 .
The resistance mediated by β-lactamases emerged rapidly, and mutations in the bla TEM , bla TEM-1 and bla SHV genes led to the emergence of novel enzymes, called extended-spectrum β-lactamases (ESBLs) due to their broad spectrum of activity, especially against oxyimino-cephalosporins 5 .
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 region 4,5 .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 ESBLproducing strains in 90% of hospitals participating in a surveillance program.These findings demonstrate that the prevalence of ESBL-producing strains varies from country to country 2,7,8 .Because of the increasing incidence of ESBL-producing Gramnegative 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.

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 techniques 9 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, respectively 9 .

Ethical considerations
Ethical approval was obtained from the Ethics Committee of the Universidade Federal do Maranhão according to the requirements of the Ministry of Health.

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 ESBLproducing 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.
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 trimethoprimsulfamethoxazole but were susceptible to amikacin, piperacillintazobactam 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 bla CTX-M gene was detected in 90.4% (104/115) of the isolates and the bla TEM gene in 75% (86/115).Seventy-five (65.2%) of the isolates carried genes encoding both the CTX-M and TEM-type enzymes (Table 3).
Although the susceptibility profile varied little between strains carrying the bla TEM and bla CTX-M genes, analysis showed that bacteria carrying the bla CTX-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 bacteria 11 .
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 Poland 14 , 7.4% in Italy 15 , 6% in Saudi Arabia 1 , 1.7% in France 16 , and 30 to 60% in Latin America 17 .These data indicate 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 studies 18 .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 countries 6,13,19,20 .
Klebsiella pneumoniae was the most frequently observed ESBLproducing microorganism in this study.Similar results have been reported in members of the family Enterobacteriaceae 7,13,18,19 and in a multicenter study investigating bacterial resistance in Brazilian hospitals 6 .In contrast, E. coli was found to be the most prevalent ESBL-producing microorganism in Saudi Arabia 1 , France 16 and Italy 15 .
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 Italy 15 .In contrast, other Brazilian studies have reported a higher prevalence of E. cloacae 13 .
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 incidence 21 .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 studies 3,13,21 , 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 urine 1,22 .However, in a study of various regions of Brazil, most ESBL-producing bacteria were isolated from blood and the respiratory tract 6 .
The main risk factors for colonization or infection with ESBLproducing bacteria are previous antibiotic use 2,5 , presence of invasive devices such as catheters 23,24 , prolonged hospital stay 25,26 , previous hospitalization 17 , delay in appropriate treatment, presence of ulcers 5,23 and ICU stay 2,17 .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 elimination 27 .
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 bacteria 6 .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 ESBLs 7 .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 types 5 .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 countries [28][29][30] .CTX-M ß-lactamases are the predominant type of ESBLs in Europe and South America 29,31 , including Brazil 32 .
High frequencies of the bla CTX-M and bla TEM genes were discovered in this study.The frequency of the bla CTX-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 bla TEM was 75% (86/115), which resembles the results of the studies conducted in Sweden and Brazil 10,33 .
A high occurrence of CTX-M-type ESBLs in E. coli has also been reported in recent studies 16,30,34,35 .However, the same was not observed for K. pneumoniae, as prevalence rates of 14.8% and 15.8% were reported in studies conducted in France 16 and Norway 35 , 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.

TABLE 4 -Detection of ESBL-coding genes by polymerase chain reaction according to species and source of infection.
+: positive, ESBL: extended spectrum β-lactamases, PCR: polymerase chain reaction.