Abstract
The aim of present work was to characterize the inhibitor-resistant TEM (IRT) β-lactamases produced by Escherichia coli in Hospital Clínico San Carlos (Madrid, Spain). Mechanisms of fluoroquinolone resistance among IRT-producing strains were also studied. Isolates with susceptibility to cephalosporins and amoxicillin–clavulanate (AMC) resistance were collected in our hospital (November 2011–July 2012) from both outpatients and hospitalized patients. Among 70 AMC-resistant E. coli strains, 28 (40%) produced IRT enzymes. Most of them were uropathogens (82.1%) and recovered from outpatients (75%). Seven different IRT enzymes were identified with TEM-30 (IRT-2) being the most prevalent, followed by TEM-40 (IRT-11). A high rate of ciprofloxacin resistance was found among IRT-producing strains (50%). Most of the ciprofloxacin-resistant isolates showed ciprofloxacin minimum inhibitory concentration >32 mg/L and contained two mutations in both gyrA and parC genes. Four IRT enzyme producers harbored the qnr gene. ST131 clone was mainly responsible for both IRT enzyme production and ciprofloxacin resistance. In conclusion, data from this study show that the frequency of IRT producers was 40% and a high rate of ciprofloxacin resistance was found among IRT-producing isolates. Current and future actions should be taken into account to avoid or reduce the development of AMC and fluoroquinolone resistance in E. coli.
Introduction
R
There are several mechanisms involved in the resistance to AMC. Inhibitor-resistant TEM (IRT) enzymes emerge through mutational events from TEM-1 or TEM-2 β-lactamases and they have decreased affinities for amino-, carboxy-, and ureidopenicillins and an altered interaction with class A β-lactamase inhibitors. 4
Classical mechanisms of quinolone resistance in E. coli are due to chromosomal mutations in the genes encoding the quinolone targets (DNA gyrase and topoisomerase IV). Mainly, the mutations are accumulated in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. 8 Resistance to quinolones can also be mediated by plasmid-borne genes such as qnr, which protect the quinolone targets from inhibition. 14
The aim of this study was to evaluate IRT β-lactamases produced by AMC-resistant E. coli isolates in Hospital Clínico San Carlos (Madrid). Since fluoroquinolones are another option for the treatment of infections caused by E. coli, our study also investigated the mechanisms involved in quinolone resistance among IRT-producing isolates. The genetic relationship between IRT producers was also determined.
Materials and Methods
Bacterial isolates
A total of 70 cefotaxime and ceftazidime susceptible but AMC-resistant E. coli strains, according to Clinical Laboratory Standards Institute (CLSI), 5 were prospectively collected from November 2011 to July 2012 in Hospital Clínico San Carlos from clinical samples of both outpatients and hospitalized patients. Fifty-six strains were isolated from urine, 7 from different exudates, 5 from surgical wound, one isolate from catheter, and one from blood. Among the 70 strains recovered, 20 (28.6%) produced nosocomial-acquired infections and 50 (71.4%) caused community-acquired infections.
Nosocomial-acquired isolates were defined as those acquired at least 48 hr after hospital admission. Community-acquired strains were those isolated in the community or within 48 hr of hospital admission.
Susceptibility testing
Bacterial identification and antibiotic susceptibility patterns were performed by using two systems: the semiautomatic Wider system (Francisco Soria Melguizo, Madrid, Spain) or VITEK®2 system (bioMérieux, Marcy-l'Etoile, France). In addition, minimum inhibitory concentrations (MICs) of AMC, piperacillin–tazobactam combinations, and ciprofloxacin were determined by Etest (bioMérieux). The results were interpreted according to CLSI guidelines. 5
IRT identification
The blaTEM gene was detected by polymerase chain reaction (PCR) and sequencing as previously described. 6 The nucleotide sequences obtained were compared with those available in the NCBI and Lahey databases.
Molecular characterization of mechanisms of resistance to fluoroquinolones
All IRT-producing isolates were screened for the presence of qnr genes (qnrA, qnrB, and qnrS) by PCR.3,10 The amplicons of those qnr-positive isolates were further sequenced. 9
Mutations in the QRDR were assessed in the IRT-producing isolates with resistance to ciprofloxacin. The gyrA and parC genes were amplified and sequenced as described elsewhere.12,15 Sequence alignments and analyses were performed online using the Basic Local Alignment Search Tools (BLAST) program.
Clonal relatedness
The genetic relationship between the IRT-producing isolates was determined by multilocus sequence typing (MLST) according to the University College University of Warwick scheme for E. coli (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). 20
Results
Identification of IRT β-lactamases and antimicrobial susceptibility
Among 70 AMC-resistant E. coli isolates, 28 were characterized as IRT producers, representing 40% of the AMC-resistant strains collected; 23 of them (82.1%) were recovered from urine, 3 (10.7%) were from exudates, and 2 (7.1%) were from other clinical samples. Twenty-one of the isolates (75%) belonged to outpatients and 7 (25%) produced nosocomial-acquired infections.
Seven different IRT β-lactamases were detected in the 28 isolates: TEM-30 (16), TEM-40 (7), TEM-31 (1), TEM-34 (1), TEM-39 (1), TEM-54 (1), and TEM-76 (1). The characteristics of IRT-producing isolates are shown in Table 1.
C, community-acquired infection; CIP, ciprofloxacin; H, nosocomial-acquired infection; IRT, inhibitor-resistant TEM; MLST, multilocus sequence typing; NAL, nalidixic acid; QRDR, quinolone resistance-determining region; SXT, trimethoprim–sulfamethoxazole.
According to the breakpoints reported by the CLSI, 5 all IRT enzyme producers were susceptible to cephalosporins, cephamycins, and carbapenems. Susceptibility to piperacilin–tazobactam was also observed for all of these strains, except one isolate. The resistance percentage of sulfamethoxazole–trimethoprim was 50%. High rates of ciprofloxacin and nalidixic acid resistance were found. Sixteen (57.1%) and 14 (50%) isolates were resistant to nalidixic acid and ciprofloxacin, respectively.
Resistance mechanisms to quinolones
All ciprofloxacin-resistant E. coli isolates contained three or four mutations in the QRDR. Table 1 shows the mutation patterns in gyrA and parC genes. Of the 14 ciprofloxacin-resistant strains, 13 had a ciprofloxacin MIC >32 mg/L and two mutations in both genes. Sequence analysis of the QRDR revealed that 10 isolates harbored the same amino acid substitution pattern (GyrA:S83L+D87N; ParC:S80I+E84V), while 3 strains carried different patterns. Two of them had the following mutations GyrA:S83L+D87T; ParC:S80I+E84V and the remaining isolate GyrA:S83L+D87N; ParC:S80I+E84G. The unique IRT-producing isolates with ciprofloxacin MIC 8 mg/L showed a single mutation in ParC (S80I) and double mutation in GyrA (S83L+D87N).
The qnr gene was detected in four IRT-producing isolates. The qnrB19 gene was identified in one TEM-34-producing strain, while the qnrS1 gene was carried by two TEM-30 and one TEM-76-producing isolates. All qnr-positive strains showed nalidixic acid and ciprofloxacin resistances and all of them had double mutation in both gyrA and parC genes.
Clonal relatedness
MLST analysis demonstrated a high degree of genetic diversity between the 28 IRT-producing isolates. Eight sequence types (STs) were identified, ST131 being the most prevalent with 14 strains (50%) followed by ST705 (5 isolates). Of these 14 isolates belonging to ST131clone, 8 produced TEM-30, 5 produced TEM-40, and only one isolate produced TEM-34. According to ciprofloxacin resistance, the susceptible isolates were genetically more diverse than the resistant isolates. Six different STs were identified in susceptible strains, while the ciprofloxacin-resistant isolates were distributed into four STs (Table 1). ST131 was the most prevalent genotype among IRT-producing strains, accounting for 50% of isolates overall and 64.3% of ciprofloxacin-resistant isolates.
Discussion
Resistance to AMC in E. coli has increased over the last years. 17 In our hospital, a high percentage of IRT-producing isolates was found. Of 70 AMC-resistant E. coli strains recovered, 40% produced IRTs. Leflon et al. in 2000 found that 41.2% of the isolates resistant to AMC exhibited the presence of an IRT pattern. 11 Conversely, lower percentages than those have been reported.13,16
Our IRT-producing isolates were mostly recovered from urine (82.1%), mainly in the community setting. Our findings are consistent with previous works that show that the most common infection between IRT producers is urinary tract infection (UTI). 13 In Spain, an increased AMC consumption at the community level has been observed. Moreover, high rates of AMC resistance can be closely related with its use. 17
Seven different IRT β-lactamases were identified in the 28 isolates, TEM-30 and TEM-40 being the most prevalent types, as previously described.13,16 Martin et al. found that a diversity of IRT enzymes could suggest an independent emergence of these enzymes and they are less transferable or selectable than extended-spectrum β-lactamases. 13 IRT-producing isolates are usually characterized as being susceptible to cephalosporins, cephamycins, and in most cases, piperacillin-tazobactam. 13 In our study, all IRT-producing strains had this susceptibility pattern, except one strain showing piperacillin–tazobactam resistance. In spite of great in vitro activity of piperacillin–tazobactam against IRT-producing isolates, no clinical information regarding its use has been plublished. 2
A high percentage of ciprofloxacin resistance was observed among our IRT producers (50%), most of them being recovered from outpatients. This percentage is higher than those described13,16 and it might be explained by antibiotic consumption. Cuevas et al. observed an increased community use of fluoroquinolones and AMC in 42 Spanish hospitals from 2001 to 2009. 7 Furthermore, the use of fluoroquinolones and AMC in outpatients may lead to the development of fluoroquinolone resistance. 7
In our study, all mutations found in gyrA and parC genes have previously been reported in fluoroquinolone-resistant E. coli.12,15,18 In general, we could also observe a relationship between the level of ciprofloxacin resistance and the number of mutations in the QRDRs as described elsewhere.1,18
Although qnr determinants confer low-level resistance on their own, it is important to detect these genes because they could contribute to select mutants with increased levels of fluoroquinolone resistance. 14 In our study, 4 IRT producers harbored the qnr gene.
Genetic characterization by MLST demonstrated that ST131 clone was the most prevalent ST among our 28 IRT-producing isolates. This genotype has been previously described among IRT producers.13,16
E. coli ST131 is a globally disseminated clone causing UTI and bacteremia 19 in both outpatients and hospitalized patients. Most of the ST131 IRT-producing isolates were recovered from outpatients with UTI, as Rogers et al. have previously reviewed. 19 This genotype is associated with several mechanisms of resistance to β-lactams, 19 including the production of IRT.13,16 Our strains belonging to this clone carried TEM-30, TEM-34, or TEM-40 enzymes.
ST131 is also characterized by producing fluoroquinolone-resistant infection. 19 In the present study, ST131 was also the most prevalent genotype among IRT-producing strains resistant to ciprofloxacin, accounting for 64.3% of the ciprofloxacin-resistant isolates. These results agree with previous reports. 1
In conclusion, the frequency of IRT enzymes among our AMC-resistant E. coli isolates was 40%, most of them being recovered from outpatients with UTI. Moreover, 50% of the IRT producers were ciprofloxacin resistant. The IRT-producing strains were genetically diverse, although approximately half of them belonged to the ST131 international clone. Current and future actions should be taken into account to avoid or reduce the development of AMC and fluoroquinolone resistance in E. coli.
Footnotes
Acknowledgments
Part of this Study was presented at the 23rd European Congress of Clinical Microbiology and Infectious Diseases (Berlin, Germany), 2013 (Poster P1181). This work was supported, in part, by a grant from the Fondo de Investigaciones Sanitarias of Spain (FIS PI13/01471).
Disclosure Statement
No competing financial interests exist.