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Journal of Clinical Microbiology, August 2006, p. 2767-2772, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.01916-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Clonality and Antimicrobial Resistance Gene Profiles of Multidrug- Resistant Salmonella enterica Serovar Infantis Isolates from Four Public Hospitals in Rio de Janeiro, Brazil

E. L. Fonseca,¹ O. L. Mykytczuk,^4,5 M. D. Asensi,¹ E. M. F. Reis,¹ L. R. Ferraz,² F. L. Paula,³ L. K. Ng,^4,5 and D. P. Rodrigues^1*

Bacteriology Department, Oswaldo Cruz Institute - FIOCRUZ, Rio de Janeiro, Brazil,¹ Public Health Laboratory, Brasilia, Brazil,² Evandro Chagas Institute, Para, Brazil,³ National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada,⁴ Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada⁵

Received 13 September 2005/ Returned for modification 7 November 2005/ Accepted 8 May 2006

Top Abstract Introduction Materials and Methods Results and Discussion References

 
In Brazil, Salmonella enterica serovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried bla_TEM, catI, ant(3")Ia and/or ant(3")Ib, sulI and/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iq cassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
For many years, ampicillin, sulfamethoxazole-trimethoprim, and chloramphenicol were the drugs of choice for the treatment of severe Salmonella infections, but increasing rates of resistance to these agents have significantly reduced their efficacies (28, 35). Subsequently, third-generation cephalosporins, due to their pharmacodynamic properties as well as low resistance levels in Salmonella, are being used to treat invasive salmonellosis (5, 11).

In 1994, Asensi and Hofer reported the presence in Rio de Janeiro, Brazil, of Salmonella enterica serovar Infantis strains that were resistant to a growing number of antimicrobial agents (6). Two years later, a nosocomial outbreak in a neonatal unit of one hospital (designated HC) was reported by De Moraes et al. (13). The authors detected multidrug-resistant serovar Infantis phenotypes, including resistance to broad-spectrum cephalosporins that was transferred by a plasmid of 148 kbp. An investigation carried out from 1998 to 1999 reported an infection due to extended-spectrum beta-lactamase (ESBL)-producing serovar Infantis in the neonatal unit of a public hospital (HC) in Rio de Janeiro, Brazil, indicating inadequate infection control practices and nursery overcrowding (30). Since then, multidrug-resistant serovar Infantis has been isolated in three other public health hospitals (designated HA, HB, and HD) of Rio de Janeiro, Brazil. Two are pediatric reference hospitals that often see children from the western and northern regions of the city, where parts of the population have lower socioeconomic and sanitary conditions. Some children were human immunodeficiency virus positive, and most suffered from recurring infections and had histories of rehospitalization. Although HC is a university-affiliated hospital and HD is a reference hospital for cancer, both provide medical care for patients with debilitating diseases such as AIDS and diabetes. In addition, these patients are subjected to prolonged hospitalizations that are often accompanied by the empirical use and sometimes overuse of antimicrobial drugs (ampicillin and/or cephalosporins and/or aminoglycosides). This led us to monitor the prevalence and antimicrobial susceptibility of serovar Infantis in hospitals in Rio de Janeiro, Brazil. The aims of this research were to (i) determine the antimicrobial susceptibility patterns, (ii) identify the main mechanisms involved in antimicrobial resistance, (iii) ascertain the presence and spread of integron-carried resistance genes, and finally, (iv) assess the macro-restriction fragment length polymorphisms between multidrug-resistant serovar Infantis strains from those hospitals.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains. Serovar Infantis strains were isolated according to the method of Costa and Hofer (12), and the antigenic characterization was based on the Kauffmann-White scheme described by Poppof (29a). This study included 35 serovar Infantis strains isolated from the stools or blood of children under 7 months who were admitted to four public hospitals (HA, HB, HC, and HD) in Rio de Janeiro, Brazil, from 1996 to 2001. Only one isolate per patient was included in the study.

Antimicrobial susceptibility testing and extended-spectrum beta-lactamase assay. Disk diffusion tests were performed according to Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) (26) recommendations by using disks (Oxoid Limited, Hampshire, England) impregnated with ampicillin (AMP; 10 µg), aztreonam (ATM; 30 µg), cephalothin (CEF; 30 µg), cefotaxime (CTX; 30 µg), ceftriaxone (CRO; 30 µg), ceftazidime (CAZ; 30 µg), cefoxitin (FOX; 30 µg), cefuroxime (CXM; 30 µg), cefepime (FEP; 30 µg), ciprofloxacin (CIP; 5 µg), chloramphenicol (CHL; 30 µg), streptomycin (STR; 10 µg), kanamycin (KAN; 10 µg), gentamicin (GEN; 10 µg), imipenem (IPM; 10 µg), nalidixic acid (NAL; 30 µg), trimethoprim-sulfamethoxazole (SXT; 25 µg), and tetracycline (TET; 30 µg). For quality control of the culture media and antimicrobial disks, Escherichia coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212, and Staphylococcus aureus ATCC 25923 were tested under the same conditions and antimicrobials as was suggested by the CLSI (26).

The method described by the CLSI for "other Enterobacteriaceae" was used to perform double-disk diffusion for the screening of ESBL-producing strains. Double-disk diffusion was performed with cephalosporin and cephalosporin/clavulanic acid combination disks (Oxoid Limited, England). Klebsiella pneumoniae ATCC 700603 (positive) and E. coli ATCC 25922 (negative) were used as control strains. In addition, the production of ESBLs in 11 serovar Infantis strains was confirmed at the National Microbiology Laboratory, Public Health Agency of Canada, by using the Mast Diagnostics ESBL detection kit (Merseyside, United Kingdom) according to the manufacturer's instructions.

Preparation of crude protein extracts and IEF. The 11 ESBL-positive isolates were grown in 2 ml of Mueller-Hinton broth at 37°C overnight, and cells were harvested by centrifugation at 16,000 x g for 2 min. After discarding the supernatant, cells were resuspended in 250 µl of 1% glycine and 30% glycerol and were sonicated twice for 30 s, with cooling of the cells on ice between sonications. Cell lysates were centrifuged at 16,000 x g for 15 min. Supernatants were collected into clean tubes and stored at –20°C. Prior to isoelectric focusing (IEF), cell extracts were tested for beta-lactamase activity by adding 50 µl of 50 µg/ml nitrocefin stock solution (Oxoid Limited, England) to 17 µl of extract and then recording the time required for the reaction to turn dark pink. The optimal reaction time was 30 to 120 s. For reaction times of 5 s or less, the extract was diluted with phosphate buffer and retested. For isolates with reaction times of 5 min or more, another extract was prepared from a culture of greater density and the test was redone. For IEF, precast polyacrylamide IEF minigels (pH 3 to 10) (Bio-Rad Laboratories, Hercules, CA) were assembled in a vertical Bio-Rad Mini-Protean II electrophoresis unit. Cathode buffer (20 mM lysine-20 mM arginine) (Bio-Rad) was added to the middle chamber, the wells were flushed, and then 10 µl of crude extract was loaded in every second well. An IEF standard with pIs ranging from 4.45 to 9.6 (Bio-Rad) was used, and a marker composed of beta-lactamases of known isoelectric points (pIs) (bla_TEM-1 [pI 5.4], bla_TEM-4 [pI 5.9], bla_TEM-3 [pI 6.3], bla_SHV-3 [pI 7.0], and bla_SHV-2 [pI 7.6]) was also used. Approximately 200 ml of anode buffer (7 mM phosphoric acid) (Bio-Rad) was added to the outer buffer chamber. The electrophoresis unit was placed on a tray and surrounded with ice. Electrophoresis was performed in three steps: 100 V for 1 h, 250 V for another hour, and finally, 500 V for 30 min. IEF gels were then dismantled from the unit, and the glass plates were separated while leaving the gel on one glass plate. To visualize beta-lactamase activity, 1 ml of nitrocefin stock solution (1 mg/ml) was added to 6 ml of molten 3% agarose in 50 mM phosphate buffer (pH 7.5) (cooled to 50 to 60°C), mixed by inversion, and then poured evenly over the gel. The presence of pink/red lines on the gel indicated beta-lactamase activity. Pictures of IEF gels were taken using a dark green filter, and the gels were transilluminated with white light.

Detection of antimicrobial resistance genes. PCR was used to detect antimicrobial resistance genes and the presence of integrons in 11 isolates resistant to (at least) the following antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole-trimethoprim, and tetracycline. Most of the primers used for the characterization of pentaresistant Salmonella serovar Typhimurium DT104 were previously described (27), and are all listed in Table 1. The DNA from the reference strains (also listed in Table 1) served as positive controls for the PCRs. Negative controls for PCRs consisted of all the reagents used for each primer pair minus the DNA template. Genomic DNA from cultures grown at 35°C on Mueller-Hinton agar with antimicrobials was extracted with a Puregene kit (Gentra Systems, Inc., Minneapolis, MN). The PCR mix for the detection of resistance genes and integrons included 1.0 µM of forward and reverse primers, 1x Taq polymerase buffer, 1.5 mM MgCl₂, 200 µM of each deoxynucleotide (dATP, dCTP, dGTP, and dTTP) (Gibco BRL, Burlington, Ontario), 0.025 U/µl Taq polymerase (Gibco BRL, Burlington, Ontario), and approximately 1 µg of template DNA. Amplification conditions for all of the PCRs, except for integron and bla_TEM amplification, were 1 cycle at 94°C for 5 min and 35 cycles for 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s. An annealing temperature of 48°C was used for the amplification of bla_TEM. Integron amplification involved 1 cycle at 94°C for 12 min and 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 5 min. PCR products were analyzed by gel electrophoresis in a 1% agarose gel run at 100 V for 1 h. To visualize band migration, the gel was stained with ethidium bromide and observed under UV light. A 100-bp or 1-kb ladder (Gibco BRL, Ontario) was used to estimate amplicon size.

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TABLE 1. PCR primers used to identify antimicrobial resistance genes and integrons in serovar Infantis

DNA sequencing. Amplicons resulting from PCRs using the primers specific to the 5' conserved and 3' semiconserved segments or universal bla_TEM primers were sequenced in both directions using an ABI Prism 377 DNA sequencer (Applied Biosystems Division of Perkin-Elmer, Foster City, CA). DNA sequences were compared to those in the GenBank database (National Center for Biotechnology Information) by using the BLAST suite of sequence similarity-searching programs (3, 4).

Pulsed-field gel electrophoresis (PFGE). Genomic DNA was prepared as described previously by Persing et al. (29) with modifications. Serovar Infantis strains were grown in 10 ml of Mueller-Hinton broth at 37°C for 12 to 18 h. Cells were harvested by centrifugation at 2,000 x g for 15 min. After discarding the supernatant, cells were resuspended with 1 ml of sterilized saline (0.85% NaCl) and the concentration was adjusted to 1 x 10⁶ cells/ml. A 5-µl aliquot of cell suspension was added to 300 µl of TEN buffer (0.5 M EDTA, 1 M Tris base, 4 M NaCl, pH 7.5) before embedding it in 340 µl of low-melting-point agarose (Sigma-Aldrich Corporation, St. Louis, MS). Plugs were subjected to lysis for 5 h at 37°C in EC buffer (0.5 M EDTA, 1 M Tris base, NaCl, N-lauryl sarcosyl, Brij 58, sodium deoxycholate, pH 7.0) (Sigma-Aldrich, MS). RNase (10 mg/ml) (Sigma-Aldrich, MS) was added to the plugs for an overnight incubation at 37°C, and then proteinase K (20 mg/ml; Gibco BRL) treatment of the plugs was performed for 24 h at 54°C. Serovar Infantis strain plugs were washed four times with CHEF-TE 1x buffer (0.5 M EDTA, 1 M Tris base, pH 7.5) (Sigma-Aldrich, MS), followed by four washes with DNS buffer (1 M Tris base, 1 M MgCl₂) (Sigma-Aldrich, MS). The digestion step was performed for 20 h at 37°C with the restriction endonuclease SpeI (10 U/µl) (Amersham Pharmacia Biotech, England). Electrophoresis was performed at 6 V/cm for 22 h with switch time intervals of 0.5 to 25 s for 19 h and 30 to 60 s for 3 h on CHEF DRIII (Bio-Rad Laboratories, Richmond, CA). The agarose gels were stained with ethidium bromide, visualized by UV transillumination, and photographed on ImageMaster VDS (Amersham Pharmacia Biotech, England). The fragment restriction patterns were analyzed by BioNumerics (Applied Maths, Belgium) and compared through the construction of a similarity matrix by using the Dice coefficient with a position tolerance setting of 1.0% and optimization setting of 1.0%, which generated a dendrogram. Serovar Branderup was included as a control. A clonal structure definition of serovar Infantis was achieved according to the criteria of Tenover et al., which correlates the number of fragment differences with genetic events (33).

Two human epidemiologically unrelated serovar Infantis strains from other public health institutions of northern (a susceptible strain from Pará) and midwestern Brazil (a multidrug-resistant strain from Brasilia) were used to assess the utility of PFGE as an epidemiological marker for nosocomial infections.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Many researchers are successfully using PFGE to investigate the epidemiologies of strains involved in outbreaks caused by beta-lactamase- and ESBL-producing bacteria (8). The PFGE analysis of the 35 serovar Infantis strains resulted in five PFGE restriction fragment profiles (Fig. 1 and 2). The comparative evaluation of the PFGE profiles yielded four fragment patterns (A1, A3, A4, and A5) for HA isolates. Three HB and five HC isolates had the PFGE profile A1 that was also encountered in 18 HA strains. The three HD strains (PFGE profile A2), isolated in 2001, showed 95% similarity to PFGE profiles A1 and A3 (Fig. 1). Macro-restriction fragment patterns of strains from northern (PFGE profile B) and midwestern (PFGE profile C) regions of Brazil were completely different from those of strains from Rio de Janeiro, Brazil.

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FIG. 1. PFGE: macro restriction fragment patterns of Salmonella serovar Infantis genome digested with SpeI. Lanes: M, molecular weight marker of Salmonella Branderup strains; A2, PFGE profile of 3 HD strains; C, PFGE profile of a midwestern hospital strain; B, PFGE profile of a northern hospital strain; A1, PFGE profile of 18 HA, 4 HB, 5 HC strains; A3, PFGE profile of 1 HA strain; A5, PFGE profile of 1 HA strain; A4, PFGE profile of 1 HA strain.

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FIG. 2. PFGE macro-restriction fragment polymorphism.

The A1 profile, found in 26 strains isolated from 1996 to 2001 in HA, HB, and HC, was considered to be the PFGE profile associated with the MDR serovar Infantis outbreaks. The PFGE patterns of serovar Infantis strains were then classified according to their similarities to the outbreak pattern. Patterns that differed from the outbreak pattern by two fragments (90%) were considered to be subtypes. A variation of two to three fragments in a PFGE profile can occur when strains are cultured repeatedly or isolated multiple times from the same patient (33). Those patterns that differed by at least four fragments were classified as unrelated types by considering that they derived from two genetic events and their isolating origins.

The susceptibility profiles of serovar Infantis are shown in Table 2. All of the strains were susceptible to carbapenem (imipenem), ciprofloxacin, nalidixic acid, and cephamycin (cefoxitin). All of the strains, except for one, were resistant to ampicillin, and most were resistant to cephalosporins (including extended spectrum). It is interesting that strains resistant to the highest number of antimicrobials (resistance profile ACSSuTTmKG, etc. [Table 2]) had similar PFGE profiles and were isolated from 1996 to 2001 from patients in different hospitals. The high prevalence of resistance to these particular antimicrobials may be due to selective pressure since these antimicrobials, with the exception of kanamycin and streptomycin, are among the agents most often prescribed in these hospitals. Resistance to kanamycin and streptomycin, however, may have been acquired through horizontal gene transfer since aminoglycoside resistance genes are often found on plasmids and transposons that encode resistance determinants for other classes of antimicrobials (34, 36). Tetracycline resistance (97.2%) and aztreonam resistance (96.1%) were also common among the multidrug-resistant strains. It is not surprising that the four hospitals involved in this study experienced great difficulties in deciding which antimicrobials to use for treatment. The implementation of effective screening methods for the detection of beta-lactamases and ESBLs as well as the establishment of surveillance programs became key factors in the control of hospital outbreaks (16).

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TABLE 2. Antimicrobial resistance and PFGE profiles for serovar Infantis strains isolated between 1996 and 2001 from four Brazilian hospitals

PCR detection of resistance genes in nine isolates resistant to five classes of antimicrobials, represented by ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, showed that all of the strains with the ACSSuTTmKG resistance profile carried bla_TEM, catI, aadA1, sulI, sulII, and tet(D) resistance genes and an integron containing an aac(6')-Iq gene cassette that codes for amikacin resistance (Table 3). The only variation among these strains was the presence or absence of the streptomycin/spectinomycin resistance gene aadA2, also known as ant(3')Ib, a variant of the gene aadA1 [ant(3')Ia]. The serovar Infantis strain with the ACSuTTmKG resistance profile was intermediately resistant to streptomycin and yet carried both aadA1 and aadA2. Redundancy of resistance genes was also detected in 10 strains carrying two sulfonamide resistance genes, sulI and sulII.

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TABLE 3. Antimicrobial resistance genes detected in multidrug-resistant serovar Infantis strains

Thirty-two (91.4%) serovar Infantis strains were classified as clavulanic-acid-inhibited ESBL-producing strains according to CLSI standards (21 from HA, 3 from HB, 5 from HC, and 3 from HD). Twenty-two strains (62.8%) were resistant to both CTX and CAZ (Table 2), which suggested the presence of at least one ESBL. According to the beta-lactamase classification scheme of Bush et al. (1995), cefotaximases are class A ESBLs (group 2be) that generally have higher hydrolytic activities against cefotaxime than ceftazidime, while ceftazidimases (also group 2be ESBLs) generally hydrolyze ceftazidime more readily than cefotaxime (10). In addition, group 2be ESBLs inactivate not only extended-spectrum cephalosporins but also monobactams such as aztreonam. Ten of the serovar Infantis strains characterized in this study were resistant to both CTX and CAZ, while only one was resistant to only CTX (Table 3). DNA sequencing of the amplicons obtained with bla_TEM primers (which targeted the conserved region of TEM-related enzymes) revealed the presence of the non-ESBL bla_TEM-1. In order to determine whether more than one beta-lactamase was produced by these 11 multidrug-resistant serovar Infantis strains, isoelectric focusing was performed (Table 4).

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TABLE 4. ß-Lactamase profiles detected in multidrug-resistant serovar Infantis strains

The pI profiles indicated the presence of beta-lactamases with pI values of 5.4, 6.3, 6.9, and 9.0. The six strains with the antibiogram ACSSuTTmKG (resistance profile, ATM, CEP, CXM, CAZ, CTX, CRO, FEP) had at least four different beta-lactamases (since there could be more than one beta-lactamase present in a strain with the same pI value), while another strain with the same resistance profile produced only two types of beta-lactamases (pIs 9.0 and 5.4). This result is significant since all seven strains are resistant to CEP, CXM, CAZ, CTX, CRO, and FEP, indicating that resistance to those cephalosporins requires the presence of only two types of beta-lactamases with pI values of 5.4 and 9.0. In addition, those seven strains are also resistant to the monobactam ATM, which indicates, according to Bush et al., that a group 2be ESBL is present within the strain (10).

The presence of identical antimicrobial resistance genes and the close relatedness of strains as determined by PFGE analysis provides evidence that the hospitals involved in this study had a salmonellosis outbreak that was caused by serovar Infantis strains that shared the same phylogenetic lineage. It is important to emphasize that strains from HC were isolated in only 1996, while strains from HB were isolated in 1997 and 1999. HA strains were isolated from 1996 to 1999. At the beginning of 2001, HD was informed about the characteristics and clonal nature of multidrug-resistant serovar Infantis so that appropriate control measures could be developed and, subsequently, serovar Infantis was no longer detected in the hospital environment. The guidelines and rules that provide for the planning of the National Program of Hospital Infection Control were defined by administrative rule GM 2.616 as of 12 May, 1998. This decree categorizes children hospitalized in high-risk nurseries as intensive-care patients requiring particular attention to infections due to multidrug-resistant pathogens (22). These patients are subjected to standard procedures for controlling nosocomial infections, such as the cleaning and disinfection of medical equipment, frequent hand washing, patient-to-patient contact precautions, and the monitoring of patients' stools for the presence of multidrug-resistant serovar Infantis. The best strategy for antimicrobial therapy and specific infection control measures for each patient was determined on a case-by-case basis (1).

The results in this study indicate that efficient surveillance programs and effective decontamination procedures must be implemented for the prevention of nosocomial outbreaks of salmonellosis caused by multidrug-resistant serovar Infantis.

 
We thank C. M. F. Reis and A. F. M. Santos (FIOCRUZ, Rio de Janeiro, Brazil) for her collaboration on the PFGE technique and photo documentation and E. Soares and his working group (FIOCRUZ, Rio de Janeiro, Brazil), who provided assistance and supplied reagents.

This work was supported by grants from the Oswaldo Cruz Institute Pos-Graduation/FIOCRUZ-Rio de Janeiro and National Council for Scientific and Technological Development (CNPq), Brazil. O. Mykytczuk's student stipend was from the National Microbiology Laboratory and the University of Manitoba, Winnipeg, Manitoba, Canada.

 
* Corresponding author. Mailing address: Laboratório de Enterobactérias, Departamento de Bacteriologia, Oswaldo Cruz Institute - FIOCRUZ, Avenida Brasil, 4365 - Pavilhão Rocha Lima, 3° andar, Manguinhos - Rio de Janeiro, Brasil 21040-361. Phone: 55 21 2598 4277. Fax: 55 21 2270 6565. E-mail: dalia{at}ioc.fiocruz.br.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Journal of Clinical Microbiology, August 2006, p. 2767-2772, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.01916-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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