Distribution of extended-spectrum β-lactamase types in a Brazilian tertiary hospital

Introduction: Epidemiological data on the prevalence of extended-spectrum β-lactamases (ESBLs) are scarce in Brazil despite the fact that these data are essential for empirical treatment and control measures. The objective of this study was to evaluate the prevalence of different ESBLs by type and distribution in a tertiary hospital in southern Brazil. Methods: We evaluated 1,827 enterobacterial isolates between August 2003 and March 2008 isolated from patients at a tertiary hospital. Samples were identifi ed using a Vitek automated system and were confi rmed by biochemical testing. The identifi ed ESBL strains were characterized by phenotypic methods, polymerase chain reaction (PCR), and sequencing. Genetic similarities were evaluated by pulsed-fi eld gel electrophoresis. Results: It was 390 (21.3%) ESBL-producing strains, which expressed the ESBLs CTX-M (292), SHV (84), CTX and SHV (10), TEM (2), and PER (2). Conclusions: The prevalence of ESBL-expressing strains was high, especially in Klebsiella pneumoniae and Enterobacter spp. CTX-M was the predominant type of ESBL observed, and its genetic variability indicates a polyclonal distribution.

Over three decades have passed since the identifi cation of the fi rst extended-spectrum β-lactamase (ESBL)-producing bacteria.Since then, the prevalence of ESBL-producing strains has increased, and new types and variants have been described.The fi rst ESBLs were derived from TEM (temoniera) and SHV (sulfhydryl-variable) β-lactamases, which are mainly found in healthcare-associated infections.Cefotaximase (CTX-M) ESBLs have increased in importance due to the increased frequency of community-acquired infections caused by strains carrying this enzyme (1) (2) .
The frequency and predominant types of ESBL vary from region to region and even between institutions within the same region.The study for monitoring antimicrobial resistance trends (SMART) surveyed ESBL-producing strains around the world and found that the CTX-M-15 variant predominates (3) .
Extended-spectrum β-lactamases are mainly found in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, but have also been described in many other species of Enterobacteriaceae and non-fermenting Gram-negative rodshaped bacteria (1) .
According to a study performed in the United States analyzing data from 2009 to 2011, North America has the lowest rates of ESBL-producers, with a prevalence of 11.4% to 16.1% in K. pneumoniae and 8.1% in E. coli.In this study, the SHV type of ESBL was most frequently observed, although the authors reported that CTX-M-14 and CTX-M-15 are increasing in prevalence and may soon replace SHV-12 as the most common ESBL types (4) .
The prevalence of the various ESBL types in Europe varies from country to country.ESBL has been found in 20.1% of E. coli (0.9%-89.7%) and 45.7% of Klebsiella spp.(2.5-100%), with greater frequencies in Mediterranean countries, Belgium, and Poland.CTX-M is the predominant type of ESBL found in Europe, and it is frequently found in community-acquired infections.Moreover, in some regions, CTX-M is more commonly found in E. coli than in K. pneumoniae (4) (5) (6) .
Epidemiological data on the frequencies of ESBLexpressing strains in Africa and Australia are rare.One study METHODS conducted in the Pacifi c region reported that 10% and 28.1% of K. pneumoniae in Australia and South Africa, respectively, are ESBL-producers.Additionally, a local study conducted in a Tunisian hospital found E. cloacae ESBL isolates that produce ESBL types CTX-M and SHV (7) (8) .
The highest prevalence of ESBL-producing strains was recorded in South America, although the frequencies vary among countries; in Guatemala, Mexico, and Peru, ESBLs are prevalent in E. coli, whereas in Brazil, Cuba, Ecuador, and Venezuela, ESBL prevalence is greater in Klebsiella spp.(i.e., ≥50%).The most common type of ESBL is CTX-M, although a few isolates harbor the TEM and SHV ESBL types.The PER-2 type, which is exclusive to South America, is the second most ESBL isolated frequently type in Argentina and has been frequently found in Uruguay, Chile, and Bolivia (9) (10) (11) .
A Brazilian survey performed as part of the SENTRY program revealed ESBL frequencies of 10% in E. coli and 50% in K. pneumoniae.Epidemiological data on the prevalence of ESBL types in Brazil are scarce and suggest that CTX-M is the predominant ESBL type.Two epidemiological studies conducted in the State of Rio de Janeiro found that the ESBL CTX-M predominates.One study found a predominance of CTX-M-2, CTX-M-59, and CTX-M-9, but not CTX-M-15 (12) .In the second study, CTX-M-15 was predominant in more than 50% of K. pneumoniae, E. coli, and E. cloacae isolates (13) .A study in Sao Paulo found 20 CTX-M-2, 14 CTX-M-59, 12 CTX-M-15, and 13 SHV genes in ESBL-expressing strains (14) .GES (Guiana Extended Spectrum β-Lactamases), BES (Brazilian Extended Spectrum β-Lactamases), and PER (Pseudomonas Extended Resistance) types have also been reported in Brazil (10) .
Most studies suggest that ESBL-producing strains are polyclonal and that the ESBL features are spread by plasmid transfer; however, during outbreaks, clonal dissemination may occur (14) (15) (16) .
The aim of this study was to investigate the prevalence of ESBL types in patients and the genetic similarities between ESBL strains in a tertiary southern Brazilian hospital.

DISCUSSION
Rev Soc Bras Med Trop 48(2):162-169, Mar-Apr, 2015 strains using a technique previously described by Kaufmann (21) and adapted by Nogueira (19) .PFGE profi les were analyzed in comparison with the standards in the Gel-Pro Analyzer 4.0 and NTSYS software to calculate the Dice similarity coeffi cient and to implement Unweighted Pair Group Method with Arithmetic Mean (UPGMA) to create dendrograms.We used the Tenover (22) criteria to defi ne a clone.
The survey of 1,827 samples yielded 483 (26.4%) strains with reduced susceptibility to third-generation cephalosporins and/or aztreonam, according to CLSI criteria (18) .Of these strains, 390 (21.3%) were ESBL-producers, including CTX-M (292), SHV (84), CTX and SHV (10), TEM (2), and PER (2).A genetic similarity analysis of CTX-M-producing strains revealed two forms of dissemination: the spread of resistant clones persisting for several years in hospitals and the transmission of resistance-encoding plasmids.This hypothesis is supported by the observation of identical clones, different types of CTX-M, and unrelated bacterial strains harboring identical ESBL genes.CTX-M-59 was an exception that was found in a single clone of E. aerogenes.ESBL clones were not restricted to certain hospital areas but were found in intensive care units (ICU) and other hospital units.They were also present as causative agents of urinary tract infections, respiratory tract infections, and bacteremia, among other diseases.Genetic similarities between CTX-M ESBL-producing K. pneumoniae, E. coli, E. cloacae, and E. aerogenes are presented in Figure 1.
The observed prevalence of ESBL-producing strains (21.3%) was similar to the prevalence reported in another Brazilian study (24.8%) based on phenotype testing.Notably, previous reports have not been conducted on the prevalence of various ESBL genotypes (23) .E. aerogenes, E. cloacae, and K. pneumoniae were the most frequently ESBL-positive species isolated.Epidemiological analysis revealed an unexpectedly high frequency of Enterobacter spp.(i.e., E. aerogenes and E. cloacae) when compared with K. pneumoniae, which is probably due to the spread of a CTX-M-59-producing E. aerogenes clone throughout the hospital.In a SENTRY study that included strains from Brazil, the authors found ESBL rates of 12.8% in Escherichia coli and 49.9% in Klebsiella spp. (11).
The CTX-M ESBL type was present in 74.8% (292/390) of all ESBL-positive strains.CTX-M was fi rst identifi ed in South America and is prevalent in Brazil.It is also the predominant ESBL type found in other regions of the world and is increasing in frequency, particularly in the context of community-acquired infections.The CTX-M-2 variant was predominant in our study and was identifi ed in 229 (58.7%) samples.CTX-M-2 was fi rst described in a strain of Salmonella isolated in Argentina and was soon reported in Brazil and other South American countries.CTX-M-2 is the predominant ESBL type in Brazil and has been found in several species of Enterobacteriaceae (11) (24) (25) .More recent studies have reported an increasing number of CTX-M-15-producing isolates (13) ( 14) (26) .
The CTX-M-59 variant found in isolates of E. aerogenes is an H89 L derivative of CTX-M-2 and was fi rst described in ESBL-producing K. pneumoniae in Brazil.CTX-M-59 has not been reported outside Brazil or in different species (24) .The bla CTX-M-2 and bla CTX-M-59 genes were identifi ed in IncA/C plasmids, reinforcing the possibility of the intraplasmid evolution of bla CTX-M-59 from bla CTX-M-2 and the implication of an effect of IncA/C on bla CTX-M dissemination (12) .CTX-M-15 is predominant in E. coli and K. pneumoniae worldwide but was found in only four of the samples in this study: E. coli (1), E. cloacae (1), E. aerogenes (1), and Serratia marcescens (1).In some countries, outbreaks of E. coli have been driven by a single ESBL-expressing clone that can be found throughout the world.CTX-M-15 was reported in Brazil for the fi rst time in 2010 (24) .Moreover, an epidemiological study from Rio de Janeiro identifi ed CTX-M-15 in more than 50% of ESBL-positive samples (13) .
The CTX-M-8 variant was found in fi ve strains of E. coli and one strain of K. pneumonia in our study.CTX-M-8 was fi rst described in Brazil in Citrobacter amalonaticus and E. cloacae; the only other report of this ESBL type was in Spain in 2009 (24) .
The CTX-M-9 variant was found in nine strains, and CTX-M-14 was found in one strain.CTX-M-9 has been reported in several Brazilian studies and is considered pandemic in Europe similarly to CTX-M-15.CTX-M-14 was fi rst reported in China and was reported in Brazil in 2010 (24) .
The SHV type was the second most common group of ESBLs observed in this study, as SHV was found in 94 (24.2%) samples.The SHV-12 variant was found in 90 (23.1%) strains.The SHV enzymes, and SHV-12 in particular, are commonly observed and have been previously reported in Brazil (14) .The SHV type is the second most common ESBL group in Brazil and other countries (16) (24) (27) .SHV-2 and SHV-27 were each found in a single (0.3%) isolate, and SHV-38 was found in two (0.6%) isolates.
The TEM type (TEM-136 variant) was observed in two (0.6%) samples.TEM ESBLs are not common in Brazil, as the only TEM type previously reported in Brazil was TEM-116 (25) .The TEM type is also less frequently observed than the CTX-M and SHV types worldwide.The PER-2 type is restricted to South America and is the second most common group in Argentina (24) .PER-2-expressing bacteria were also described during an outbreak in Uruguay.In Brazil, the PER-2 type is infrequent and was observed in two isolates previously reported by our group (28) .
Ten isolates produced two types of ESBL: SHV-12 and CTX-M-2.ESBL was also associated with narrow-spectrum β-lactamases (non-ESBL TEM and SHV) in 204 samples.These associations are very common; moreover, different β-lactamases can be encoded on the same plasmid (29) .
The high genetic variability observed among ESBLproducing bacteria indicates polyclonal spread to K. pneumoniae, E. cloacae, and E. coli and a high rate of transfer of ESBL genes between bacteria in healthcare-associated infections.Other Brazilian studies have found similar results (13) (30) .Polyclonal dissemination is also supported by the incidence of identical clones with different types of CTX-M ESBLs as well as by unrelated isolates expressing identical enzymes.CTX-M-59 did not disseminate and was expressed by a single clone of Enterobacter aerogenes.Other instances of ESBL clonal dissemination were associated with outbreaks in specifi c hospital wards (31) (32) .By the time of preparation of this manuscript, we have not found an association between the clonal spread of E. aerogenes and an outbreak.ESBL-producing isolates did not remain restricted to a single ward.Another Brazilian study found similar results regarding K. pneumonia ESBL (25) .The β-lactamase CTX-M has spread horizontally and vertically, and clones were not restricted to certain hospital areas but were present in ICUs and other in-patient units.This information is of paramount importance for nosocomial infection control.
Klebsiella pneumoniae isolates consisted of two clonal groups subdivided into groups A1-A15 (related) and group B. Clonal group A contains CTX-M-2 and CTX-M-9.Clone A K. pneumoniae was found throughout the study period in several clinical isolates, suggesting that Clone A Klebsiella pneumoniae is an endemic microorganism in the hospital.Clone B was present from 2004 to 2008 in different departments, primarily in intensive care units.The other K. pneumoniae isolates exhibited similarities below the level of highly related or related samples.
The dissemination of ESBLs in E. coli was mostly polyclonal with small highly related clonal groups designated A, B, C, and D. Clone A was found in urine samples of three hematology patients in 2008, with one isolate producing CTX-M-15 and two isolates producing CTX-M-2.Clone B appeared in samples from 2007 and 2008 and included CTX-M-2 and CTX-M-8.Clone C was present in samples from 2004 to 2008; four ICU isolates expressed CTX-M-2, and two ESBL subclones, CTX-M-2 and CTX-M-8, were isolated in other wards.Clone D consisted of thre representative isolates from different wards and different years.
E. aerogenes clones producing CTX-M-59 were isolated from 2004 to 2008, mainly from blood and urine samples obtained from patients in the ICU.Only two isolates showed no clonal relationship with the other isolates.
The spread of E. cloacae was mostly polyclonal, with fi ve main groups, each including more than three similar strains.Clonal groups were designated A, B, C, D, and E, all of which expressed the ESBL CTX-M-2.Except for clone E, which was mainly isolated from blood samples, the remaining clones were predominantly isolated from urine.High rates of resistance to cephalosporins and aztreonam were observed; however, a few samples were susceptible according to CLSI criteria (18) .Thus, the clinical effi cacy of cephalosporin and aztreonam against ESBL-producing bacteria remains uncertain.Cefepime susceptible-dose dependence (SDD) was found in 12.8% of the ESBL isolates; in cases such as these, higher doses are recommended for infected patients (18) .
Carbapenems are considered the drug of choice for treating infections by ESBL-producing microorganisms.This study showed good sensitivity of isolates to carbapenems (99.3% for imipenem, 98.2% for meropenem, and 86.4% for ertapenem), but resistant isolates were also observed (mainly ertapenem).ESBLs do not degrade carbapenems effi ciently but can confer resistance when associated with reduced permeability of the membrane to the drug.Carbapenems displayed good potency and activity, reinforcing the fact that they are an important therapeutic option against these microorganisms.Aminoglycosides have also been associated with high sensitivity rates but are also associated with high toxicity and limited indications.The carbapenems are the most widely used antimicrobial agents to treat infections caused by ESBL-producing strains (9) (10) .However, the indications for their use should be clearly defi ned because the indiscriminate use of carbapenems has led to the emergence of resistant strains (30) .
The clinical isolates analyzed in our study were susceptible to tigecycline but to a lesser degree than they were to carbapenems.Tigecycline use was approved by the Food and Drug Administration for intra-abdominal infections, skin and soft tissue infections, and community-acquired pneumonia.
Tigecycline has not been approved for the treatment of bacteremia and urinary tract infections; additional studies are needed to demonstrate its effi cacy (30) .
ESBLs are increasing in frequency among several species of enterobacteria.This information is of paramount importance for nosocomial infection control.CTX-M was the predominant type observed in our study, which is consistent with other reports from South America.The ESBL CTX-M has been found in hospitals and in community, animal, and environmental samples, which illustrates the challenge associated with containing bacteria expressing this ESBL type.The diversity of species in which CTX-M was found and the great genetic variability observed among E. coli, K. pneumoniae, and E. cloacae ESBL-producing bacteria indicate a predominantly polyclonal spread and high transfer effi ciency of ESBL genes between bacteria in the hospital environment.Moreover, the spread of resistance can occur through the transmission of resistant strains, and resistant clones can be established and remain in hospitals for several years, eventually spreading to other hospitals and causing various types of infections.One example is the CTX-M-59 gene expressed in an E. aerogenes strain, which has not been found in other species and has exhibited monoclonal spread.Epidemiological evidence and local studies are important to elucidate the spread of EBSL types, which present a worldwide public health problem.
Clone A was isolated in 2004-2007, clone B was isolated in 2005-2006, C and E were isolated in 2005-2007, and D was isolated in 2004-2006.All clones were found in different clinics suggesting that they were spread across various hospital departments.