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

Abstract

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 profiles 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 profile 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 identified 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 findings 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.

Class 1 integron; Klebsiella pneumonia ; Phylogenetic groups


INTRODUCTION

Klebsiella pneumoniae is a bacillus that is commonly associated with serious nosocomial infections, such as septicemia, pneumonia, urinary tract infections, and meningitis11. Minarini LA, Gales AC, Palazzo IC, Darini Al. Prevalence of community-occurring extended spectrum beta-lactamase-producing Enterobacteriaceae in Brazil. Curr Microbiol 2007; 54:335-341.33. Tu YC, Lu MC, Chiang MK, Huang SP, Peng HL, Chang HY, et al. Genetic requirements for Klebsiella pneumoniae induced liver abscess in oral infection model. Infec Immu 2009; 77:2657-2671.. This species is classified into three phylogenetic groups, KpI, KpII, and KpIII, based on nucleotide variations in the constitutively expressed genes gyrA, parC, and rpoB44. Brisse S, Van Himbergen T, Kusters K, Verhoef J. Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clin Microbiol Infec 2004; 10:942-945.,55. Fevre C, Passet V, Weil FX, Grimont PAD, Brisse S. Variants of the Klebsiella pneumoniae OKP chromosomal beta-lactamase are divided into two main groups, OKP-A and OKP-B. Antimicrob Agents Chemother 2005; 49:5149-5152.. Mobile genetic elements, such as integrons, contribute to the evolution and dissemination of multidrug resistance genes (blaCTX-M, blaIMP, and blaGES) in K. pneumoniae through horizontal or vertical transfer66. Chen L, Chavda KD, Fraimow HS, Mediavilla JR, Melano RG, Jacobs MR, et al. Complete nucleotide sequences of blaKPC-4- and blaKPC-5-harboring IncN and IncX plasmids from Klebsiella pneumoniae strains isolated in New Jersey. Antimicrob Agents Chemother 2013; 57:269-276.99. Bebrone C, Bogaerts P, Delbrück H, Bennink S, Kupper MB, Rezende de Castro R, et al. GES-18, a new carbapenem-hydrolyzing GES-type β-Lactamase from Pseudomonas aeruginosa that contains Ile80 and Ser170 Residues. Antimicrob Agents Chemother 2013; 57:396-401.. 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, respectively1010. Levesque C, Piche L, Larose C, Roy PH. PCR mapping of ntegrons reveals several novel combinations of resistence genes. Antimicrob Agents Chemother 1995; 39:185-191.1212. Martínez N, Mendonza MC, Rodriguez I, Soto S, Bances M, Rodicio R. Detailed structure of integrons and transposons carried by large conjugative plasmids responsible for multidrug resistance in diverse genomic types of Salmonella enterica serovar Brandenburg. J Antimicrob Chemother 2007; 60:1227-1234.. These genetic elements can be found in different species of Gram-negative bacteria1313. Ahangarzadeh Rezaee M, Langarizadeh N, Aghazadeh M. First report of class 1 and class 2 integrons in multidrug-resistant Klebsiella pneumoniae isolates from northwest Iran. Jpn J Infect Dis 2012; 65:256-259.1717. Taherikalani M, Maleki A, Sadeghifard N, Mohammadzadeh D, Soroush S, Asadollahi P, et al. Dissemination of class 1, 2 and 3 integrons among different multidrug resistant isolates of Acinetobacter baumannii in Tehran hospitals, Iran. Pol J Microbiol 2011; 60:169-174. from hospital environments. Based on the amino acid sequence of the IntI protein, five classes of mobile integrons have been described1818. Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet 2010; 44:141-166.. Classes 1, 2, and 3 are the most commonly detected1919. Stalder T, Barraud O, Casellas M, Dagot C, Ploy MC. Integron Involvement in Environmental Spread of Antibiotic Resistance. Frontiers Microbiol 2012; 3:119.. 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. pneumoniae66. Chen L, Chavda KD, Fraimow HS, Mediavilla JR, Melano RG, Jacobs MR, et al. Complete nucleotide sequences of blaKPC-4- and blaKPC-5-harboring IncN and IncX plasmids from Klebsiella pneumoniae strains isolated in New Jersey. Antimicrob Agents Chemother 2013; 57:269-276.,1313. Ahangarzadeh Rezaee M, Langarizadeh N, Aghazadeh M. First report of class 1 and class 2 integrons in multidrug-resistant Klebsiella pneumoniae isolates from northwest Iran. Jpn J Infect Dis 2012; 65:256-259.,1616. Ibrahim N, Wajidi MF, Yusof MY, Tay ST. The integron prevalence of extended-spectrum beta-lactamase producing enterobacterial isolates in a Malaysian teaching hospital. Trop Biomed 2011; 28:668-671.,2020. Chagas TP, Alves RM, Vallim DC, Seki LM, Campos LC, Asensi MD. Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil. Braz J Infect Dis 2011; 15:420-425.,2121. Mobarak-Qamsari M, Ashayeri-Panah M, Eftekhar F, Feizabadi MM. Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae. Braz J Microbiol 2014; 44:849-854.. 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. pneumoniae2121. Mobarak-Qamsari M, Ashayeri-Panah M, Eftekhar F, Feizabadi MM. Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae. Braz J Microbiol 2014; 44:849-854.,2222. Bhattacharjee A, Sen MR, Prakash P, Gaur A, Anupurba S, Nath G. Observation on integron carriage among clinical isolates of Klebsiella pneumoniae producing extended spectrum β-lactamases. Ind J Med Microbiol 2010; 28:207-210..

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 2323. Vinué L, Sáenz Y, Somalo S, Escudero E, Moreno MA, Ruiz-Larrea F, et al. Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain. J Antimicrob Chemother 2008; 62:934-937.. 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 profiles displayed by the isolates.

METHODS

Bacterial isolates

A total of 51 K. pneumoniae isolates already typed for phylogenetic groups2424. Melo MES, Cabral AB, Maciel MAV, Silveira VM, Lopes ACS. Phylogenetic Groups among Klebsiella pneumoniae isolates from Brazil: Relationship with antimicrobial resistance and origin. Current Microbiology 2011; 62:1596-1601. 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 37°C for 18h in brain-heart infusion broth (BHI) or Luria-Bertani broth (LB).

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

Antibiotic susceptibility analysis

Antibiotic susceptibility was tested on Mueller-Hinton agar using the disk diffusion method Clinical and Laboratory Standards Institute (CLSI)2525. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. 23th informational supplement. CLSI M100-S23. Wayne, PA: CLSI; 2013.. The following antibiotics were tested: amoxicillin-clavulanate (AMC), amoxicillin (AMO), amikacin (AMI), ampicillin (AMP), aztreonam (ATM), ceftazidime (CAZ), cefotaxime (CTX), cefepime (CPM ), ciprofloxacin (CIP), chloramphenicol (CLO), streptomycin (EST), gentamicin (GEN), imipenem (IPM), kanamycin (KAN), nalidixic acid (NAL), sulfamethoprim (TSP), and tetracycline (TET).

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 -20°C until use.

Detection of class 1 integrons through PCR

Amplification reactions were prepared in a total volume of 25µl containing 1ng of genomic DNA, 2 units of Taq DNA polymerase (Promega), 200µM of each deoxyribonucleotide triphosphate (dNTP) (Promega), 1.5mM MgCl2, 1µM of each primer (3′CS (AAGCAGACTTGACCTGAT) and 5′CS (GGCATCCAAGCAGCAAG), and 1X reaction buffer2626. Bissonnette L, Roy PH. Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. J bacteriol 1992; 174:1248-1257.. The cycling conditions were one cycle of 5 min at 95 °C; 30 cycles of 1 min at 95°C, 1min at 65°C, and 1min at 72°C; and one cycle of 10min at 72°C. The amplification products were visualized in 1% agarose gels in Tris-borate buffer (TBE) buffer.

RESULTS

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).

Antimicrobial resistance

The identified K. pneumoniae isolates with class 1 integrons exhibited the following high levels of resistance to antibiotics: AMP 100% (n=19); AMO 57.9% (n=11); AMC 42.1% (n=8); CTX 94.7% (n=18); CAZ 73.7% (n=14); CPM 47.4% (n=9); and IPM and meropenem 36.8% (n=7). In addition, resistance to sulfamethoxazole associated with trimethoprim was observed in 94.7% (n=18) of these isolates, and resistance to monobactam aztreonam (ATM) was observed in 73.7% (n=14) of these isolates. The K. pneumoniae isolates were more susceptible to aminoglycosides, fluoroquinolones, and CLO than to penicillin, cephalosporin, sulfa, or monobactams. Eighteen (94.7%) of the K. pneumoniae isolates carrying class 1 integrons exhibited simultaneous sulfamethoxazole/trimethoprim resistance. K10-R was the only isolate with one class 1 integron and no resistance to sulfamethoxazole/trimethoprim. Conversely, the K12-A and K20-P isolates did not contain class 1 integrons but displayed resistance to sulfamethoxazole/trimethoprim (Table 1).

DISCUSSION

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 profiles of the studied isolates. Few studies have evaluated the presence of class 1 integrons in K. pneumoniae isolates from the Brazilian northeast2727. Lopes ACS, Veras DL, Lima AMS, Melo RCA, Ayala J. blaCTX-M-2; and bla CTX-M-28 extended-spectrum β-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil. Mem Inst Oswaldo Cruz 2010; 105:163-167.. Chagas et al.2020. Chagas TP, Alves RM, Vallim DC, Seki LM, Campos LC, Asensi MD. Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil. Braz J Infect Dis 2011; 15:420-425. showed that all K. pneumoniae ESBL-producing isolates from Rio de Janeiro and São Paulo, Brazil carried class 1 integrons. Ahangarzadeh Rezaee et al.1313. Ahangarzadeh Rezaee M, Langarizadeh N, Aghazadeh M. First report of class 1 and class 2 integrons in multidrug-resistant Klebsiella pneumoniae isolates from northwest Iran. Jpn J Infect Dis 2012; 65:256-259. 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 identified in clinical isolates only; these results are similar to those reported by Dalsgaard et al.1111. Dalsgaard A, Forslund A, Petersen A, Brown DJ, Dias F, Monteiro S, et al. Class 1 Integron-Borne, Multiple-Antibiotic Resistance Encoded by a 150-Kilobase Conjugative Plasmid in Epidemic Vibrio cholerae O1 Strains Isolated in Guinea-Bissau. J Clin Microbiol 2000; 38:3774-3779. and confirm 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.2828. Penteado AP, Castanheira M, Pignatari ACC, Guimarães T, Mamizuka EM, Gales AC. Dissemination of blaIMP-1- carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23. Diagn Microbiol Infect Dis 2009; 63:87-91. and Lopes et al.2727. Lopes ACS, Veras DL, Lima AMS, Melo RCA, Ayala J. blaCTX-M-2; and bla CTX-M-28 extended-spectrum β-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil. Mem Inst Oswaldo Cruz 2010; 105:163-167., 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 confirmed 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 resistance2929. Agersø Y, Peirano G, Aarestrup FM. dfrA25, a novel trimethoprim resistance gene from Salmonella agona isolated from a human urine sample in Brazil. J Antimicrob Chemother 2006; 58:1044-1047.,3030. Beutlich J, Rodriguez I, Schroeter A, Kasbohrer A, Helmuth R, Guerra B. A predominant multidrug-resistant Salmonella enteric seroval saintpaul clonal line in German Turkey and related food products. Appl Environ Microbiol 2010; 76:3657-3667.. 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 findings3131. Grape M, Sundström L, Kronvall G. Two new dfr genes in trimethoprim-resistant integron-negative Escherichia coli isolates. Antimicrob Agents Chemother 2007; 51:1863-1864.3434. Toro M, Rojo-Bezares B, Vinue' L, Undabeitia E, Torres C, Yolanda S. In vivo selection of aac(6′)-Ib-cr and mutations in the gyrA gene in a clinical qnrS1-positive Salmonella enterica serovar Typhimurium DT104B strain recovered after fluoroquinolone treatment. J Antimicrob Chemother 2010; 65: 1945-1949.. 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 group44. Brisse S, Van Himbergen T, Kusters K, Verhoef J. Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates. Clin Microbiol Infec 2004; 10:942-945.. 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 years3535. Cabral AB, Melo RCA, Maciel MAV, Lopes ACS. Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil. Rev Soc Bras Med Trop 2012; 45:572-578.. 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.

REFERENCES

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    Levesque C, Piche L, Larose C, Roy PH. PCR mapping of ntegrons reveals several novel combinations of resistence genes. Antimicrob Agents Chemother 1995; 39:185-191.
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    Martínez N, Mendonza MC, Rodriguez I, Soto S, Bances M, Rodicio R. Detailed structure of integrons and transposons carried by large conjugative plasmids responsible for multidrug resistance in diverse genomic types of Salmonella enterica serovar Brandenburg. J Antimicrob Chemother 2007; 60:1227-1234.
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    Ahangarzadeh Rezaee M, Langarizadeh N, Aghazadeh M. First report of class 1 and class 2 integrons in multidrug-resistant Klebsiella pneumoniae isolates from northwest Iran. Jpn J Infect Dis 2012; 65:256-259.
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    Taherikalani M, Maleki A, Sadeghifard N, Mohammadzadeh D, Soroush S, Asadollahi P, et al. Dissemination of class 1, 2 and 3 integrons among different multidrug resistant isolates of Acinetobacter baumannii in Tehran hospitals, Iran. Pol J Microbiol 2011; 60:169-174.
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    Chagas TP, Alves RM, Vallim DC, Seki LM, Campos LC, Asensi MD. Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil. Braz J Infect Dis 2011; 15:420-425.
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    Mobarak-Qamsari M, Ashayeri-Panah M, Eftekhar F, Feizabadi MM. Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae. Braz J Microbiol 2014; 44:849-854.
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    Bhattacharjee A, Sen MR, Prakash P, Gaur A, Anupurba S, Nath G. Observation on integron carriage among clinical isolates of Klebsiella pneumoniae producing extended spectrum β-lactamases. Ind J Med Microbiol 2010; 28:207-210.
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    Vinué L, Sáenz Y, Somalo S, Escudero E, Moreno MA, Ruiz-Larrea F, et al. Prevalence and diversity of integrons and associated resistance genes in faecal Escherichia coli isolates of healthy humans in Spain. J Antimicrob Chemother 2008; 62:934-937.
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    Lopes ACS, Veras DL, Lima AMS, Melo RCA, Ayala J. blaCTX-M-2; and bla CTX-M-28 extended-spectrum β-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil. Mem Inst Oswaldo Cruz 2010; 105:163-167.
  • 28
    Penteado AP, Castanheira M, Pignatari ACC, Guimarães T, Mamizuka EM, Gales AC. Dissemination of blaIMP-1- carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23. Diagn Microbiol Infect Dis 2009; 63:87-91.
  • 29
    Agersø Y, Peirano G, Aarestrup FM. dfrA25, a novel trimethoprim resistance gene from Salmonella agona isolated from a human urine sample in Brazil. J Antimicrob Chemother 2006; 58:1044-1047.
  • 30
    Beutlich J, Rodriguez I, Schroeter A, Kasbohrer A, Helmuth R, Guerra B. A predominant multidrug-resistant Salmonella enteric seroval saintpaul clonal line in German Turkey and related food products. Appl Environ Microbiol 2010; 76:3657-3667.
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  • 35
    Cabral AB, Melo RCA, Maciel MAV, Lopes ACS. Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil. Rev Soc Bras Med Trop 2012; 45:572-578.
  • FINANCIAL SUPPORTFundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for fi nancial support.

Publication Dates

  • Publication in this collection
    Mar-Apr 2014

History

  • Received
    31 Jan 2014
  • Accepted
    11 Apr 2014
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