Molecular epidemiology of cefotaxime-resistant but ceftazidime-susceptible Enterobacterales and evaluation of the in vitro bactericidal activity of ceftazidime and cefepime.

Extended-spectrum ß-lactamases' (ESBLs) production is the main resistance mechanism to third-generation cephalosporins (TGCs) in gram-negative bacilli. In Argentina, there is a high prevalence of cefotaximase-type ESBLs (CTX-M). For this reason, dissociated resistance phenotype (DRP) displaying a profile of resistance to cefotaxime (CTX) and susceptibility to ceftazidime (CAZ) might be detected. The aims of this study were to determine the prevalence of DRP in Enterobacterales clinical isolates, to characterize the mechanisms responsible for this phenotype and to evaluate the in vitro behaviour against different antibiotics. Sixty Enterobacterales resistant to any TGC were studied, and among them, 25% displayed a DRP. The ß-lactamases associated with DRP were 5/11 CTX-M-2, 4/11 CTX-M-14, 1/11 CTX-M-15 and 1/11 CMY-2 in E. coli, 2/3 CTX-M-2 and 1/3 CMY-2 in P. mirabilis and 1/1 CTX-M-14 in K. pneumoniae. Furthermore, CTX-M-2 and CTX-M-14 were related with DRP in both wild-type isolates and the corresponding transconjugants. Time-kill experiments showed CAZ bactericidal activity on CTX-M-2-and CTX-M-14-producing strains and bacterial regrowth in those CMY-2 producers. An opposite behaviour was evident when cefepime (FEP) was used. However, CAZ and gentamicin combination showed a synergistic effect against the CMY-2 producers. We concluded that Enterobacterales with DRP responded differently to CAZ or FEP depending on the type of ß-lactamase they possess, suggesting that these cephalosporins could be a therapeutic option. Therefore, the characterization of the involved resistance mechanism might contribute to define the appropriate antibiotic treatment.

Fast and easy detection of CMY-2 in Escherichia coli by direct MALDI-TOF mass spectrometry.

Fast typing methods for third generation cephalosporin resistance mechanisms are needed to guide appropriate treatment and prevent potential dissemination events. In this study we used a novel short and fast methodology for the identification of CMY-2 in 50 well characterized clinical isolates of E. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry- MALDI-TOF MS. Samples were prepared using the double layer sinapinic acid technique for detection of intact proteins Comparison among mass spectral profile of different strains between m/z 35,000-45,000 Da showed that two groups of isolates could be differentiated after peak analysis. A single distinctive peak with different intensities, at approximately m/z 39,800 Da was found in all CMY-2 producing strains (transconjugant, transformant and wild type) and consistently absent in the control groups (ESBL producers and susceptible strains). Statistical results showed 100% values for sensitivity and specificity, indicating a perfect test and a high discriminative power. In this study, we demonstrated that MALDI-TOF MS has the potential to detect directly the most clinically relevant acquired AmpC ß-lactamase, the CMY-2-enzyme, in E. coli with a less time-consuming process as compared to conventional methods. Our results may constitute the basis for further research to detect other ß-lactamases, or even other resistance markers.

Susceptibility to ß-lactams and quinolones of Enterobacteriaceae isolated from urinary tract infections in outpatients.

The antibiotic susceptibility profile was evaluated in 71 Enterobacteriaceae isolates obtained from outpatient urine cultures in July 2010 from two health institutions in Santa Fe, Argentina. The highest rates of antibiotic resistance were observed for ampicillin (AMP) (69%), trimethoprim/sulfamethoxazole (TMS) (33%), and ciprofloxacin (CIP) (25%). Meanwhile, 21% of the isolates were resistant to three or more tested antibiotics families. Thirty integron-containing bacteria (42.3%) were detected, and a strong association with TMS resistance was found. Third generation cephalosporin resistance was detected in only one Escherichia coli isolate, and it was characterized as a blaCMY-2 carrier. No plasmid-mediated quinolone resistance (PMQR) was found. Resistance to fluoroquinolone in the isolates was due to alterations in QRDR regions. Two mutations in GyrA (S83L, D87N) and one in ParC (S80I) were observed in all CIP-resistant E. coli. It was determined to be the main phylogenetic groups in E. coli isolates. Minimum Inhibitory Concentration (MIC) values against nalidixic acid (NAL), levofloxacin (LEV), and CIP were determined for 63 uropathogenic E. coli isolates as MIC50 of 4 µg/mL, 0.03125 µg/mL, and 0.03125 µg/mL, respectively, while the MIC90 values of the antibiotics were determined as 1024 µg/mL, 64 µg/mL, and 16 µg/mL, respectively. An association between the phylogenetic groups, A and B1 with fluoroquinolone resistance was observed. These results point to the importance of awareness of the potential risk associated with empirical treatment with both the families of antibiotics.

Susceptibility to β-lactams and quinolones of Enterobacteriaceae isolated from urinary tract infections in outpatients

Abstract The antibiotic susceptibility profile was evaluated in 71 Enterobacteriaceae isolates obtained from outpatient urine cultures in July 2010 from two health institutions in Santa Fe, Argentina. The highest rates of antibiotic resistance were observed for ampicillin (AMP) (69%), trimethoprim/sulfamethoxazole (TMS) (33%), and ciprofloxacin (CIP) (25%). Meanwhile, 21% of the isolates were resistant to three or more tested antibiotics families. Thirty integron-containing bacteria (42.3%) were detected, and a strong association with TMS resistance was found. Third generation cephalosporin resistance was detected in only one Escherichia coli isolate, and it was characterized as a blaCMY-2 carrier. No plasmid-mediated quinolone resistance (PMQR) was found. Resistance to fluoroquinolone in the isolates was due to alterations in QRDR regions. Two mutations in GyrA (S83L, D87N) and one in ParC (S80I) were observed in all CIP-resistant E. coli. It was determined to be the main phylogenetic groups in E. coli isolates. Minimum Inhibitory Concentration (MIC) values against nalidixic acid (NAL), levofloxacin (LEV), and CIP were determined for 63 uropathogenic E. coli isolates as MIC50 of 4 μg/mL, 0.03125 μg/mL, and 0.03125 μg/mL, respectively, while the MIC90 values of the antibiotics were determined as 1024 μg/mL, 64 μg/mL, and 16 μg/mL, respectively. An association between the phylogenetic groups, A and B1 with fluoroquinolone resistance was observed. These results point to the importance of awareness of the potential risk associated with empirical treatment with both the families of antibiotics.