Prevalence and Characterization of Plasmid-Mediated Quinolone Resistance Genes in Extended-Spectrum β-Lactamase–Producing Enterobacteriaceae Isolates in Mexico

Clinical isolates included in the study

This study evaluated 226 ESBL-producing Enterobacteriaceae isolates collected from eight hospitals in five regions of Mexico: northwest (Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado [ISTE-S]), Sonora; west (Hospital Civil de Guadalajara [HCG-J]), Jalisco; center (Instituto Nacional de Cancerología [INCan-DF], and Sanatorio Durango [SD-DF]), México Distrito Federal; south (Hospital General de Acapulco [CGA-G]), Guerrero; and north (Hospital de Altas Especialidades No. 25 del IMSS [HAE-NL], Hospital San Juan Tecnológico [HSJT-NL], and Centro Regional de Control de Enfermedades Infecciosas [CRCEI]), Monterrey Nuevo León. Only one isolate was permitted per patient and the culture sources included blood, urine, secretion, catheter, and other sites. All isolates were sent to the National Institute of Public Health in Morelos, Mexico, where identification was confirmed and all data were integrated into a centralized database.

Three bacterial species were included: ESBL-producing Escherichia coli (136 isolates), ESBL-producing K. pneumoniae (56 isolates), and ESBL-producing Enterobacter cloacae (34 isolates). All isolates were collected between March 2005 and June 2009. The distribution of the isolates according to the hospital is shown in Table 1. The ESBL production was detected in E. coli and K. pneumoniae isolates according to the Clinical and Laboratory Standards Institute (CLSI) methodology (M100-S21). ⁷ Cefotaxime (30 μg), cefotaxime–clavulanic acid (30/10 μg), ceftazidime (30 μg), and ceftazidime–clavulanic acid (30/10 μg) discs were used. An increase of >5 mm in the inhibition zone of the combination disc compared with the cephalosporin-only disc suggested an ESBL-producing organism.

Screening of PMQR genes in clinical isolates

The qnrA, qnrB, qnrS, qnrC, and qnrD genes were screened by separated multiplex polymerase chain reaction (PCR) ⁵ and confirmed by single PCR, and the qepA and aac(6′)-Ib-cr genes were analyzed by single PCR with specific primers for each gene (Appendix Table 1). The aac(6′)-Ib-cr gene was differentiated from the wild-type gene by PCR digestion with BstI5 enzyme and confirmed by nucleotide sequence. The qepA gene was amplified using a 1× enhancer buffer (Invitrogen). All PCR products were purified by means of High-Pure PCR Product Purification Kit (Roche Applied Science), sequenced using chain termination method with a Big-Dye Terminator kit (Applied Biosystems), and analyzed on an ABIPRISMA 3100 (Applied Biosystems). The nucleotide sequences were compared on GenBank database by means of BLASTx searches.

Analysis for qnr-positive isolates

The β-lactamase genes encoding for ESBL bla_CTX-M, bla_SHV, and bla_TLA were screened by PCR using specific primers (Appendix Table 1). The minimum inhibitory concentrations (MICs) against nalidixic acid, ciprofloxacin, levofloxacin, cefotaxime, ceftazidime, and gentamicin were determined by agar dilution plate method following the CLSI and were interpreted according to CLSI performance standard M100-S21. ⁷ The antimicrobials were supplied and stored according to the manufacturer's instructions. E. coli 25922 was used as reference strain for susceptibility testing.

PCR amplification of the quinolone resistance–determining regions (QRDRs) of the gyrA and parC genes (except parC of E. cloacae) were performed with specific primers (Appendix Table 1). Both strands of the purified PCR products were sequenced and the DNA sequences were analyzed by BLASTx for the identification of QRDRs of GyrA and ParC protein and were compared with the protein sequences of K. pneumoniae and E. coli GyrA and ParC proteins deposited in the GenBank, respectively.

The quinolone resistance transfer genes were assayed by mating experiments, as described by Miller, ¹⁸ in 21 qnr-positive isolates, which were selected according to the hospital of origin and clonal relationship. Conjugation experiments were performed using azide-resistant E. coli J53, and transconjugants were selected on LB plates containing sodium azide (100 μg/ml); nalidixic acid (8 μg/ml) or ampicillin (100 μg/ml) and cefotaxime (1 μg/ml) were used for counterselection of plasmid-mediated quinolone and cephalosporin resistance. The phenotype resistance transferred was determined using LB agar plates with eight different types of antibiotics: nalidixic acid (8 μg/ml), ciprofloxacin (0.5 μg/ml), ampicillin (100 μg/ml), cefotaxime (1 μg/ml), tetracycline (15 μg/ml), chloramphenicol (10 μg/ml), kanamycin (25 μg/ml), and gentamicin (16 μg/ml). The confirmation of ESBL production and PMQR genes was carried out as described earlier. The incompatibility groups of plasmids were analyzed in all transconjugants by PCR-based replicon typing. ⁴

Plasmid DNA was extracted from clinical isolates according to the method described by Kieser. ¹⁵ Plasmids R1 (92 kb), R6K (40 kb), RP4 (54 kb), R1 (205 kb), and pUA21 (300 kb) were used as molecular size markers.

All qnr-carrying isolates were analyzed by pulsed-field gel electrophoresis (PFGE), ³¹ and DNA was obtained as previously described by Kaufmann. ¹⁴ Isolates from each bacterial species were analyzed using the GelCompar II software (Applied Math). The similarity percentage was determined in a dendrogram derived from UPGMA and Dice coefficients (band position tolerance and optimization were set at 0.7% and 0.65%, respectively).

Epidemiological and prevalence data of the ESBL-producing Enterobacteriaceae isolates

The age of the patients from whom the isolates were collected was between 18 and 74 years; 73.4% (36/49) of the isolates corresponded to male patients and 24.6% (13/49) to female patients. The sites of the samples are listed as follows: blood 78 (34.5%), urine 61 (27%), secretion 30 (13.2%), catheter 3 (1.3%), and other sites 54 (24%).

The prevalence of qnr determinants was 21.6% (49/226) and it was distributed as follows: 55.8% (19/34) among E. cloacae isolates, 50% (28/56) among K. pneumoniae isolates, and 1.4% (2/136) among E. coli isolates (Table 1). Among these 49 qnr-carrying isolates, 71.4% (35/49) carried qnrB; qnrB6 type turned out to be the most prevalent (37.1%, 13/35), followed by qnrB2 (28.5%, 10/35) and qnrB1 and qnrB5 (17.1%, 6/35). The qnrS1 gene was identified in 24.4% (12/49) of the isolates, and qnrA1 in 18.3% (9/49). The qnrC and qnrD genes were not identified. Seven isolates (14.2%) carried two alleles of qnr genes. Three of them carried A1/S1 genes; another three carried A1/B genes and one isolate carried B/S1 genes (Table 2). The prevalence of the aac(6′)-Ib-cr gene was 49.5% (112/226); from these isolates, 18 of 112 (16%) contained both wild-type and mutant genes. The distribution of this gene is given as follows: 61.7% (84/136) of the isolates corresponded to E. coli, 33.9% (19/56) to K. pneumoniae, and 26.4% (9/34) to E. cloacae (Table 1).

Characteristics of qnr-positive isolates

Among the qnr-positive isolates, 76.3%, 74.6%, and 74.6% were resistant to nalidixic acid, ciprofloxacin, and levofloxacin, respectively. Thirty-three of 49 qnr-positive isolates (67.3%) were nalidixic acid resistant, with MICs >512 mg/L. Thirty isolates (61.2%) were ciprofloxacin resistant, with MICs >64 mg/L. Thirty-two isolates (69.3%) were levofloxacin resistant, with MICs ranging from 16 to >64 mg/L. The remaining isolates were susceptible or intermediate to nalidixic acid (23.7%), ciprofloxacin (25.4%), and levofloxacin (25.4%), according to the CLSI. Regarding gentamicin, 64.4% of the isolates were resistant to this antibiotic (mainly >64 μg/ml). In terms of cephalosporin antibiotics, such as cefotaxime and ceftazidime, 98.3% of the isolates were resistant to both antibiotics (Table 2). In these qnr-positive isolates, the prevalence of aac(6′)-Ib-cr gene was 55.1% (27/49); 20% (10/49) of these isolates contained both wild-type and mutant genes. The distribution was as follows: 60.7% (17/28) of the isolates corresponded to K. pneumoniae; 36.8% (7/19) to E. cloacae, and 100% (2/2) to E. coli isolates (Table 2). In the case of transconjugants containing both the qnrA1 and the aac(6′)-Ib-cr genes (TK06268), the level of resistance to ciprofloxacin was 1.0 μg/ml, a value close to the clinical breakpoint for susceptibility. However, a onefold increase in the MIC for nalidixic acid was observed in the transconjugants that transferred only the aac(6′)-Ib-cr genes (Table 3).

The ESBL SHV-type was the most prevalent (81.6%, 40/49), and it was distributed as follows: 55% (22/40) corresponded to SHV-12; 27.5% (11/40) corresponded to SHV-5; 15% (6/40) to SHV-2a, and 2.5% (1/40) to SHV-2. The ESBL CTX-M-type was identified in 44.8% (22/49) of the isolates: 95% (21/22) corresponded to CTX-M-15 and 5% (1/22) to CTX-M-1; 8.1% (4/49) corresponded to TLA-1, considering that the 40.8% of the isolates contains two or three ESBLs. The prevalence of the ESBL SHV-CTX-M-type was 32.6% (15/49), followed by CTX-M-TLA-1 (6.1%, 3/49) and SHV-CTX-M-TLA-1 (2%, 1/49) (Table 2). The ESBL CTX-M-type was identified in all cases (with the exception of three isolates) in combination with an SHV-type. The K01254 isolate and its respective transconjugant (TK01254) showed the ESBL-producing phenotype only in the case of cefotaxime, and both strains were susceptible in vitro (Tables 2 and 3).

The prevalence of mutations in the gyrA and parC genes was 69.3% and 80%, respectively (parC gene from E. cloacae was not analyzed). The most frequent mutations identified were the Ser83Ile and Ser80Ile for GyrA in 55.1% (34/49) of the isolates and the ParC in 90% (27/30) (Table 2).

Regarding the 21 mating experiments, 7 were successful: 3 of them transferred the cefotaxime and nalidixic acid resistance phenotype (A) and 4 transferred only the cefotaxime resistance phenotype (B). The qnrB alleles were preferably cotransferred with the SHV-type, whereas the qnrA1 was cotransfered with the CTX-M-15 and aac(6′)-Ib-cr genes. For aac(6′)-Ib-cr, only one transconjugant turned out to be positive (Table 3). The transferred plasmids belonged to different incompatibility groups (IncN, IncF_rep, and IncFIA) (Table 3). All transconjugants showed from two- to threefold increase in the MIC of nalidixic acid, with respect to E. coli J53.

Plasmid DNA was extracted from all the qnr-carrying isolates. All isolates contained from one to four plasmids, within a range of <40 to 330 kb in size. In general, the plasmid profiles were heterogeneous; nevertheless, in related clones from each hospital (clones A, B, and C), the profiles were homogeneous, including the nonrelated clones from the same hospital (hospital 4). The qnrB-type determinants were detected in four transconjugants (Table 3) with aac(6′)-Ib-cr gene on a 80-kb plasmid. The qnrS1 and qepA genes were transferred into 170- and 150-kb plasmids, respectively (Table 3). The incompatibility groups of the plasmid encoding qnrA, aac(6′)-Ib-cr, and qepA1 were determined; however, the plasmids encoding the qnrB-type genes turned out to be negative by PCR-based replicon typing.

The genotyping analysis among qnr-positive isolates showed genetic relationship between the isolates from each hospital, and it pointed out to the presence of endemic clones at the four hospitals that rendered qnr-positive isolates.

Characteristics of qepA-positive isolates

The qepA gene corresponded to qepA1 allele and its prevalence was 1.7% (4/226) (Table 1). This gene was identified in four qnr-negative E. coli isolates from different hospitals (isolates 03210 and 03212 corresponded to hospital 1 and isolates 09220 and 10246 corresponded to hospitals 3 and 8, respectively) (Table 1); however, all isolates were obtained in 2008. These E. coli isolates showed a multidrug resistance pattern with the following MICs: nalidixic acid (>256 μg/ml), ciprofloxacin (>64 μg/ml), levofloxacin (64 μg/ml), gentamicin (1 μg/ml), cefotaxime (≥256 μg/ml), and ceftazidime (512 μg/ml).

The CTX-M-15 corresponded to ESBL encoded in three qepA-positive isolates and SHV-12 was identified in the other one. The 09220 isolate contained the 150- and 110-kb plasmids, and the transfer of cefotaxime and ampicillin resistance (phenotype C) corresponded to 150-kb plasmid with IncFIA and IncF_rep incompatibility groups. This plasmid encoded the qepA1 and CTX-M-15 and was negative for aac(6′)-Ib-cr gene (Table 3). Onefold increase in the MIC for nalidixic acid was observed in this transconjugant (Table 3).