Characterization of Enterobacteriaceae Isolates Obtained from a Tertiary Care Hospital in Mexico,Which Produces Extended-Spectrum β-Lactamase

Setting and clinical isolates

This study was performed at the Hospital Civil de Guadalajara “Fray Antonio Alcalde,” a 1000-bed tertiary care teaching hospital. The hospital provides services to the city of Guadalajara (Jalisco, Mexico), with approximately 30,000 admissions annually. The hospital includes adult and pediatric beds that are distributed between 31 wards and four separate buildings. Adult wards are in buildings 1 and 2, pediatric wards are in building 2, obstetrics, gynecology, the women's surgical ward, and the neonatal intensive care wards are in building 3, and building 4 houses the emergency pediatric ward and the pediatric intensive care unit.

During the 6-month study period (from October 2010 to March 2011), all E. coli (n=460) and K. pneumoniae (n=78) isolates obtained from clinical specimens were analyzed. These isolates were identified using Sensititre plates (TREK Diagnostic Systems, West Sussex, England) incubated at 36°C for 18–24 hr. Using a Sensititre Auto Reader, data for each plate were analyzed and recorded. Only readings reported by the software to be excellent or good were accepted. Bacterial isolates were stored in the Brucella broth containing 15% glycerol at −70°C.

Antimicrobial susceptibility and the ESBL phenotype

The minimum inhibitory concentration (MIC) for all isolates was determined using broth microdilutions applied to Sensititre plates, as described in the guidelines published by the Clinical and Laboratory Standards Institute (CLSI). ⁶ The antimicrobial panel assay included amikacin (AMK), ampicillin (AMP), amoxicillin/clavulanic acid (AMX), aztreonam (AZT), cefepime (FEP), ceftazidime (CAZ), cefotaxime (CTX), cefuroxime (CXM), ceftriaxone (CRO), cephalotin (CEF), chloramphenicol (CHL), ciprofloxacin (CIP), fosfomycin (FOS), gentamicin (GEN), imipenem (IPN), levofloxacin (LVX), meropenem (MEM), nitrofurantoin (NIT), norfloxacin (NOR), ticarcillin (TIC), trimethoprim/sulfametoxazol (SXT), and tobramycin (TOB). Multidrug resistance (MDR) was further defined as resistance to three or more classes of antimicrobials using the following class definitions: penicillins (AMP), cephalosporins (FEP, CAZ, CTX, CXM, CRO, CEF), carbapenems (IPN, MEM), fluoroquinolones (LVX, CIP), aminoglycosides (AMK, GEN, TOB), and tetracyclines (TET).

The combination disk method, with ceftazidime and cefotaxime applied individually and in combination with inhibitory clavulanic acid, was also used to detect expression of ESBL according to the guidelines of the CLSI. ⁶

PCR screening and characterization of ESBLs

CTX-, SHV-, and TLA-type β-lactamases were screened using PCR. Briefly, template DNA was prepared from two fresh colonies inoculated into 100 μl distilled water. Cells were subsequently lysed at 95°C for 10 min. Cellular debris was removed with centrifugation (15,000 g for 2 min) and supernatants were diluted 1:10 in distilled water for use as template DNA. PCR amplification of each gene was performed using the following oligonucleotides: for bla_CTX-M, CTX-MF, 5′-GCTGTTGTTAGGAAGTGTG-3′ and CTX-MR, 5′-GGTGACGATTTTAGCCGCC-3′ ¹⁶ ; for bla_SHV-type, SHV-F5′-GGGTTATTCTTATTTGTCGC-3′ and SHV-R5′-TTAGCGTTGCCAGTGCTC-3′ ¹⁸ ; and for bla_TLA-1, TLA-1F, 5′-TCTCAGCGCAAATCCGCG-3′ and TLA-1R, 5′-CTATTTCCCATCCTTAACTA-3′. ¹⁶ For each 50 μl PCR amplification, 5 μl of heat-extracted template DNA, 10 pmol of each primer, 1× reaction buffer, 2 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate, and 1.5 U Taq DNA polymerase were included. The PCR conditions included a denaturation step (5 min at 94°C), followed by 25 cycles of 30 sec at 94°C, 30 sec at 58°C, and 30 sec at 72°C, and a final extension at 72°C for 7 min. In all cases, the resulting PCR products were electrophoresed in 1.5% agarose gels, then individual, distinct product bands were purified using a High Pure™ PCR Purification Kit (Boheringer).

For bla_SHV, sequencing was performed at the Instituto de Biotecnología (Universidad Nacional Autonoma de Mexico). For CTX-M, sequencing was performed using the dideoxy chain termination method and an automatic sequencer (ABI PRISM 377-18 kit EL: Taq FS Dye Terminator Cycle Sequencing Fluorescence-Based Sequencing). Primers used for PCR amplification were also used for sequencing of amplified PCR products. Amino acid sequences were obtained using the Translate tool available at ExPASy (www.expasy.ch/tools/dna). Multiple alignments of nucleotide and amino acid sequences were performed using ClustalW software (http://clustalw.genome.jp/), and sequences were compared to GenBank and Lahey databases (www.ncbi.nlm.nih.gov/ and www.lahey.org/Studies/, respectively).

Pulsed-field gel electrophoresis

All ESBL isolates were genotyped using PFGE. ⁸ Briefly, genomic DNA was isolated in an agarose-embedded form and was subjected to enzymatic digestion with 50 U XbaI. Agarose digests were then placed in preformed wells of an agarose gel and separated by electrophoresis in the 0.5× TBE buffer using a CHEF Mapper system (Bio-Rad Laboratories, Inc.). After 23 h, gels were stained with ethidium bromide and PFGE profiles were visualized under UV light. Patterns were interpreted using Tenover criteria, ¹⁹ and isolates exhibiting >60% similarity were considered genetically related.

Plasmid profile

Plasmids were extracted from isolates using the method of Kieser et al. ⁹ and were vertically electrophoresed in 0.7% agarose gels stained with ethidium bromide. Plasmids R6K (40 kb), RP4 (54 kb), R1 (205 kb), and pUA21 (300 kb) were used as molecular size markers.

Isolates, prevalence of ESBLs, and antimicrobial susceptibility

Between October 2010 and March 2011, a total of 75 E. coli and 21 K. pneumoniae ESBL-producing isolates were identified, representing a prevalence of 16.3% and 26.9%, respectively. The clinical characteristics of these isolates are listed in Table 1. The in vitro susceptibility of these isolates against 22 antimicrobial agents is listed in Table 2. All of the isolates from both ESBL species were found to be MDR, according to the criteria of Magiorakos et al. ¹¹ Moreover, for the E. coli isolates, >95% susceptibility was demonstrated for imipenem, meropenem, amikacin, fosfomycin, and nitrofurantoin. K. pneumoniae isolates exhibited a similar susceptibility, in addition to being susceptible to gentamicin. Susceptibility to nitrofurantoin was also observed for 33.3% of K. pneumoniae isolates, while <6% of the isolates were susceptible to cefuroxime and ticarcillin.

Clonal relatedness, molecular characterization of ESBL-producing isolates, and plasmid patterns

PFGE analysis was performed for both E. coli and K. pneumoniae isolates. In the former group, 11 clusters were detected: C (n=3), D (n=2), E (n=3), F (n=3), G (n=2), H (n=2), I (n=2), J (n=2), L (n=2), M (n=2), and O (n=2). In the latter group, only 3 clusters were detected: A (n=5), B (n=5), and K (n=2) (Table 1). Based on this profiling, 68/75 E. coli isolates and 18/21 K. pneumoniae isolates were selected for further molecular characterization.

In PCR assays, an 810 bp bla_CTX-M-type product was obtained from 85% (64/75) of E. coli isolates and from 76% (16/21) of K. pneumoniae isolates (Table 1). Of these isolates, 25 and 7 PCR products, respectively, were completely sequenced. All of the amino acid sequences obtained were homologous with CTX-M-15 ESBL. For the bla_SHV-type gene, a 928 bp band was obtained from 17% (13/75) and 86% (18/21) of E. coli and K. pneumoniae isolates, respectively (Table 1). Sequencing of these isolates produced amino acid sequences that were homologous with SHV-5 ESBL. In contrast, the bla_TLA-1gene was not detected in any of the 96 isolates analyzed. Furthermore, a majority of the ESBL-producing isolates analyzed were found to harbor between one and five different plasmids that were up to 300 kb in length.