Investigation of class 1 integrons in Klebsiella pneumoniae clinical and microbiota isolates belonging to different phylogenetic groups in Recife , State of Pernambuco

Introduction: The high prevalence of Klebsiella pneumoniae infections is related to the ability of K. pneumoniae to acquire and disseminate exogenous genes associated with mobile elements, such as R plasmids, transposons and integrons. This study investigated the presence of class 1 integrons in clinical and microbiota isolates of K. pneumoniae belonging to different phylogenetic groups and correlated these results with the antimicrobial resistance profi les of the studied isolates. Methods: Of the 51 isolates of K. pneumoniae selected for this study, 29 were from multidrug-resistant clinical isolates, and 22 were from children’s microbiota. The susceptibility profi le was determined using the disk diffusion method, and class 1 integrons were detected through polymerase chain reaction (PCR). Results: The results showed that none of the 22 microbiota isolates carried class 1 integrons. Among the 29 clinical isolates, 19 (65.5%) contained class 1 integrons, and resistance to sulfamethoxazole/ trimethoprim was identifi ed in 18 of these isolates (94.7%). Among the K. pneumoniae isolates with class 1 integrons, 47% belonged to the KpI phylogenetic group, and one isolate (14.3%) carrying these genetic elements belonged to the KpIII group. Conclusions: The wide variety of detected class 1 integrons supports the presence of high rates of antimicrobial resistance, genetic variability, and rapid dissemination of beta-lactamase genes among K. pneumoniae clinical isolates in recent years in hospitals in Recife-PE, Brazil. The fi ndings of this study indicate that the surveillance of K. pneumoniae integrons in clinical isolates could be useful for monitoring the spread of antibiotic resistance genes in the hospital environment.

Klebsiella pneumoniae is a bacillus that is commonly associated with serious nosocomial infections, such as septicemia, pneumonia, urinary tract infections, and meningitis [1][2][3] .This species is classifi ed into three phylogenetic groups, KpI, KpII, and KpIII, based on nucleotide variations in the constitutively expressed genes gyrA, parC, and rpoB 4,5 .Mobile genetic elements, such as integrons, contribute to the evolution and dissemination of multidrug resistance genes (bla CTX-M, bla IMP, and bla GES ) in K. pneumoniae through horizontal or vertical transfer [6][7][8][9] .The mobile class 1 integrons are predominantly found in clinical isolates and are associated with transposon Tn21.Class 1 integrons are composed of two conserved regions, a 3´ conserved segment (3´ CS) and a 5´ conserved segment (5´ CS), as well as an internal variable region containing gene cassettes that encode antimicrobial resistance determinants.The 3´ CS segment contains the qacE Δ 1 and sul 1 genes, which confer resistance to ethidium bromide and quaternary ammonium compounds and to sulfonamide, respectively [10][11][12] .These genetic elements can be found in different species of Gram-negative bacteria [13][14][15][16][17] from hospital environments.Based on the amino acid sequence of the IntI protein, fi ve classes of mobile integrons have been described 18 .Classes 1, 2, and 3 are the most commonly detected 19 .Class 1 integrons are the most widespread and have been frequently found in extended-spectrum β-lactamase (ESBL)producing clinical isolates of Enterobacteriaceae, including K. pneumoniae 6,13,16,20,21 .Class 2 integrons occur less frequently in ESBL-producing Escherichia coli and K. pneumoniae, and class 3 integrons are rarely found in ESBL-producing K. pneumoniae 21,22 .
Class 1 and class 2 integrons have also been found in Escherichia coli from fecal samples from healthy individuals in Spain, indicating that individuals from the community may serve as reservoirs for these genetic elements 23 .Nevertheless, no published study has correlated the presence of these genetic elements with K. pneumoniae phylogenetic groups or with microbiota isolates.Thus, the present study evaluated the presence of class 1 integrons in clinical and microbiota isolates of K. pneumoniae from different phylogenetic groups in Recife, Brazil, and it correlated their presence with the antimicrobial resistance profi les displayed by the isolates.

Bacterial isolates
A total of 51 K. pneumoniae isolates already typed for phylogenetic groups 24 were analyzed for the presence of class 1 integrons.Twenty-nine of these isolates were from hospitalized patients from the City of Recife, Pernambuco, Brazil, and 22 were from normal oropharyngeal and fecal microbiota from healthy 3-to 4-year-old children attending a day care center called Lar Fabiano de Cristo in the Várzea district of Recife, Brazil (Table 1).All the cultures were stored in 20% glycerol at -70°C and were grown at 37ºC for 18h in brain-heart infusion broth (BHI) or Luria-Bertani broth (LB).

Genomic DNA extraction
Genomic deoxyribonucleic acid (DNA) was extracted from direct colony suspensions in 200µl of distilled water.The suspensions were heated at 100°C for 10min and centrifuged (5min/10,000xg), and 100µl of the recovered supernatant was stored at -20ºC until use.

Class 1 integrons
Among the 51 K. pneumoniae clinical and microbiota isolates analyzed in this work, 19 (37.2%) carried class 1 integrons.Nineteen (65.5%) of the 29 clinical isolates carried class 1 integrons, as indicated by amplicons ranging from 750 to >2,080bp.None of the 22 microbiota isolates carried these genetic elements (Table 1).Three clinical isolates (K5A, K10P, and K14P) contained two integrons each, with amplicon sizes ranging from 750 to >2,080bp.The examination of the relationship between the phylogenetic groups and the presence of class 1 integrons revealed that 16 (80%) of the clinical isolates carrying class 1 integrons belonged to the KpI phylogenetic group.Clinical isolates from 1998 and 1999 exhibited lower integron frequencies compared with those from 2007 and 2008 (Table 1).
This study investigated the presence of class 1 integrons in clinical and microbiota isolates of K. pneumoniae from different phylogenetic groups and correlated these results with the antimicrobial resistance profi les of the studied isolates.Few studies have evaluated the presence of class 1 integrons in K. pneumoniae isolates from the Brazilian northeast 27 .Chagas et al. 20 showed that all K. pneumoniae ESBL-producing isolates from Rio de Janeiro and São Paulo, Brazil carried class 1 integrons.Ahangarzadeh Rezaee et al. 13 found that integrons were widely prevalent in clinical isolates of K. pneumoniae from northwestern Iran and were associated with multidrug resistance.
In the present study, class 1 integrons were identifi ed in clinical isolates only; these results are similar to those reported by Dalsgaard et al. 11 and confi rm that integrons are predominantly found in clinical isolates.
Because no integrons were identified in the studied K. pneumoniae microbiota isolates from healthy individuals, we suggest that they are not reservoirs for class 1 integrons.Thus, K. pneumoniae clinical isolates are the main reservoirs for these genetic elements and disseminate them to other bacterial species through horizontal or vertical transfer in the hospital environment.Most of the clinical isolates carried one integron; however, the K5A, K10P, and K14P isolates each contained two integrons.This result is consistent with reports by Penteado et al. 28 and Lopes et al. 27 , who observed additional integrons in isolates and suggested the presence of high diversity in these genetic elements in K. pneumoniae isolates from Recife.The 3´ segment of class 1 integrons contains the sul 1 gene,  which encodes sulfonamide resistance.The K10-R isolate carried a class 1 integron but did not exhibit sulfamethoxazole/ trimethoprim resistance.The most likely reason for this result is that this isolate does not express the sul1 gene.Our study confi rmed a correlation between sulfamethoxazole/trimethoprim resistance and the presence of class 1 integrons: 94.7% of the analyzed isolates carried class 1 integrons and exhibited sulfamethoxazole/trimethoprim resistance 29,30 .The K12-A and K20-P isolates did not carry class 1 integrons but displayed sulfamethoxazole/trimethoprim resistance.The sul1 gene encodes resistance to sulfonamides only, and we evaluated the associated sulfamethoxazole/trimethoprim.The most common mechanism of trimethoprim resistance involves variants of the dihydrofolate reductase (DFR), and the absence of class 1 integrons in these two isolates suggests that they may express the dfrA gene and that this gene could be located in other genetic elements, such as plasmids, transposons and chromosomes, which could explain our fi ndings [31][32][33][34] .We observed that isolates belonging to the KpI phylogenetic group had the highest frequency of class 1 integrons; some class 1 integrons were observed in isolates from the KpII group, but very few were observed in isolates from the KpIII group.This high frequency of class 1 integrons is one of the factors explaining why isolates from the KpI group presented the highest level of resistance, followed by isolates from the KpII group and isolates from the KpIII group 4 .We also observed that the frequency and diversity of class 1 integrons in the K. pneumoniae clinical isolates in Recife increased between 1998 and 1999 and between 2007 and 2008.This result is consistent with the observed rapid dissemination of beta-lactamase genes along with resistance to extended-spectrum cephalosporins and carbapenems among K. pneumoniae clinical isolates in Recife in recent years 35 .In this study, the diversity of class 1 integrons in K. pneumoniae isolates from Recife, Brazil favors the dissemination of resistance among K. pneumoniae and other species in the hospital environment.Moreover, this study also indicates that the surveillance of K. pneumoniae integrons in clinical isolates could be useful for monitoring the spread of antibiotic resistance genes in the hospital environment.

FINANCIAL SUPPORT
Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for fi nancial support.

TABLE 1 -
Specimens, origins, phylogenetic groups, presence of class 1 integrons, and antimicrobial resistance profi les of Klebsiella pneumoniae isolates from Recife, Brazil.