MICs Of Ticarcillin Research Articles

Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins.

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.

First Outbreak of Klebsiella pneumoniae Clinical Isolates Producing GES-5 and SHV-12 Extended-Spectrum β-Lactamases in Korea

Klebsiella pneumoniae clinical isolates producing extended-spectrum β-lactamases (ESBLs; TEM and SHV types) are frequently implicated in hospital outbreaks. There are also reports of K. pneumoniae isolates producing various non-TEM, non-SHV ESBLs: CTX-M and GES/IBC types (1). Presently, the different GES-type ESBLs are designated by identical names (8). To clarify the misleading nomenclature of GES-type ESBLs, we propose maintaining the current denomination concerning the fully characterized GES-3 and GES-4 ESBLs reported by Wachino et al. (12, 13) and renaming the variants (GES-3 and GES-4) reported by Vourli et al. (11) as GES-5 and GES-6, respectively. In 2004, six K. pneumoniae clinical isolates producing ESBLs were collected from different patients and distributed among several wards (intensive care unit, neurosurgery, pulmonary internal medicine, and general medicine) at Bundang CHA Medical Center, Republic of Korea. These isolates were identified from sputum samples (for three isolates), urine (for one isolate), bile (for one isolate), and pus (for one isolate). Antibiotic susceptibility testing by disk diffusion tests that were performed according to the recommendations of the Clinical and Laboratory Standards Institute (2) with BBL (Cockeysville, Md.) disks and by the double-disk synergy test of Jarlier et al. (4) suggested the presence of ESBL(s). Double-disk synergies were observed for amoxicillin-clavulanic acid (CLA), ceftazidime, cefotaxime, and aztreonam. All isolates were resistant to penicillins, ceftazidime, cefotaxime, aztreonam, tobramycin, gentamicin, and trimethoprim-sulfamethoxazole. They were susceptible to cefepime and represented reduced susceptibility to imipenem. The analysis of genomic DNA, digested with SpeI and resolved by pulsed-field gel electrophoresis (PFGE) (5), revealed the same macrorestriction pattern among all isolates, which were therefore classified as indistinguishable. Screening for ESBL gene(s) was performed by PCR using the primers for blaTEM, blaSHV, blaGES, blaPER, blaVEB, blaTOHO, blaSFO, blaBES, blaFEC, blaCME, blaTLA, blaCTX-M, and blaOXA (1, 5, 7, 10). PCR amplification experiments using primers designed to amplify GES- and SHV-type ESBL genes gave only positive results for all isolates. The nucleotide sequence of the respective amplicon for the blaSHV gene revealed 100% identity with blaSHV-12 from K. pneumoniae (6). That for the blaGES gene differs by only one silent mutation (G→A) at position 54 from blaGES-5, described elsewhere for Escherichia coli (11). On the isoelectric focusing gel, two β-lactamase activities with pIs of 5.8 and 8.2 were detected in all isolates. On the basis of DNA sequencing and pI values, the β-lactamase activity of pI 8.2 corresponds to that of SHV-12 β-lactamase, and the pI value of 5.8 represents GES-5 β-lactamase. Six isolates were used as donors in transconjugation experiments. Ceftazidime-resistant transconjugants were obtained in all cases at frequencies ranging from 10−4 to 10−5 per donor cell. All transconjugants produced only GES-5. The SHV-12 gene was not cotransferred, indicating that the bla genes for GES-5 and SHV-12 resided on different genetic elements. All GES-5-producing transconjugants were resistant to penicillins and ceftazidime and susceptible to cefoxitin, cefotaxime, and aztreonam. This phenotype is characteristic of GES/IBC-producing Enterobacteriaceae (9-13). All transconjugants were susceptible to cefepime and showed reduced susceptibilities to imipenem. MICs of ticarcillin, ceftazidime, and cefotaxime were reduced by clavulanic acid (Table ​(Table1).1). This resistance was due to the acquisition of large (>70-kb) plasmids that exhibited identical HindIII restriction patterns and harbored the GES-5 ESBL gene. Due to the presence of SHV-12 in all isolates, the MIC patterns of clinical isolates were different from those of their transconjugants. TABLE 1. MICs of β-lactams for ESBL-producing K. pneumoniae clinical isolates, their transconjugants, and the azide-resistant E. coli J53 recipient The carbapenemase bioassay (the modified cloverleaf test) (5) showed that extracts derived from all transconjugants producing GES-5 supported growth of the indicator, E. coli ATCC 25922, in the presence of an imipenem disk (30 μg), resembling the effect of OXA-23, which was used as a positive control. In contrast, cell extracts of a ceftazidime-sensitive K. pneumoniae clinical isolate collected from the Bundang CHA Medical Center did not cause any visible inhibition of carbapenemase activity. These findings indicated that the reduced susceptibilities of all isolates and their transconjugants to imipenem were due to production of GES-5 ESBL with the detectable carbapenemase activity. Finding the same macrorestriction pattern by PFGE indicated that an endemic K. pneumoniae strain producing GES-5 and SHV-12 ESBLs was present in different wards at the Bundang CHA Medical Center in 2004. Six GES-5-producing transconjugants were also resistant to tobramycin, gentamicin, and trimethoprim-sulfamethoxazole. Although the complete nucleotide sequences of integrons from six clinical isolates were not determined, screening for the presence of class 1 through 4 integrase genes from large plasmids by PCR with primer pairs previously described (3) revealed the presence of class 1 integrons. The complete class 1 integron mapping of large plasmids from six clinical isolates and the possible spread of the GES-5 ESBL gene to other Korean hospitals are currently under investigation.

In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy

We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene. Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC). Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection. This case illustrates how strains of P. aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations.

Extended-spectrum β-lactamase-producing strain of Acinetobacter baumannii isolated from a patient in France

Sir, Acinetobacter spp., especially Acinetobacter baumannii, are common opportunistic pathogens in immunocompromised patients and currently cause 10% of nosocomial infections in intensive care unit patients. They tend to be resistant to multiple antibiotics and to produce cephalosporinases. Indeed, hyperproduction of cephalosporinases, together with decreased outer membrane permeability, are the predominant mechanisms of resistance to ceftazidime amongst A. baumannii isolates. Most extended-spectrum -lactamases (ESBLs) are the result of mutations that alter the hydrolytic properties of the restricted spectrum penicillinases, TEM-1 and -2 and SHV-1. These enzymes are principally mediated by plasmids which spread readily amongst Enterobacteriaceae and their presence is detected by the demonstration of synergy between clavulanic acid, which inhibits the lactamase, and a third-generation cephalosporin (most effectively ceftazidime) with the double-disc diffusion test. In the course of routinely assessing all ceftazidime-resistant Acinetobacter spp. isolates by the double-disc diffusion test, we identified a strain of A. baumannii (Ama-1) exhibiting only slight synergy that was most evident when the ceftazidime disc was placed 2 cm from the clavulanic acid disc. The MICs of amoxycillin, ticarcillin, piperacillin, ceftazidime and imipenem for the isolate were determined by the agar dilution method according to recommendations of a working party of the British Society for Antimicrobial Chemotherapy. Ama-1 was resistant to all of the -lactams tested, with the exception of imipenem. In the presence of clavulanic acid at a fixed concentration of 2 mg/L, the MIC of ticarcillin was reduced from 512 mg/L to 256 mg/L and that of ceftazidime from 512 mg/L to 128 mg/L. Similarly, in the presence of sulbactam at a concentration of 4 mg/L, the MIC of ceftazidime fell from 512 mg/L to 256 mg/L. The activity of any -lactamase(s) produced by this isolate is therefore inhibited only minimally by either -lactamase inhibitor. In an attempt to determine the molecular basis of this resistance phenotype, we assayed for a putative ESBL gene by PCR analysis with TEM-specific primers (5 -GTATGGATCCTCAACATTTCCGTGTCG-3 and 5 -ACCAAAGCTTAATCAGTGAGGCA-3 ) and SHVspecific primers (5 -TTATCTCCCTGTTAGCCACC-3 and 5 -GATTTGCTGATTTCGCCG-3 ). Neither gene was demonstrated. However, primers used to detect the gene for the ESBL, PER-1 (5 -ATGAATGTCATTATAAAAGC-3 and 5 -AATTTGGGCTTAGGGCAGAA-3 ), yielded a 925 bp PCR product. The sequence analysis of this product revealed total identity with blaPER-1 which was originally detected in a strain of Pseudomonas aeruginosa. A survey recently carried out in Turkey showed that 46% of Acinetobacter spp. hospital isolates possessed PER1-type -lactamases. In this study, attempts to demonstrate plasmid carriage of blaPER-1 in Ama-1 and to transfer the ESBL resistance phenotype from Ama-1 to Escherichia coli DH10B by mating-out assays and by electroporation (electrotransformation) were unsuccessful. Ama-1 was isolated following culture of a rectal swab obtained from a 90 year old female patient on the intensive care unit. She had previously been admitted to two other hospitals in Paris but, to the best of our knowledge, had not travelled to Turkey, nor had she been in contact with either Turkish patients or travellers to Turkey. PER-1-positive A. baumannii strains were not isolated from the rectal swabs of 16 other patients on the intensive care unit at the same time as the patient from whom Ama-1 was recovered. Moreover, comparison of ApaI-digested genomic DNA extracted from Ama-1 with DNA from four randomly selected PER-1-type -lactamase-positive and one PER1-type -lactamase-negative A. baumannii strains from Turkey by pulsed-field gel electrophoresis (PFGE) revealed different restriction patterns (Figure). Thus, the French isolate was not clonally related to the Turkish strains. This is the first report from outside Turkey of the detection of an ESBL in an Acinetobacter spp. clinical isolate. We believe that it is also the first description of the sequencing-based identification of an ESBL gene in an

Clavulanic acid in combination with ticarcillin: an in-vitro comparison with other β-lactams

The in-vitro activity of ticarcillin in combination with clavulanic acid was compared with that of ticarcillin alone, piperacillin, cefotaxime and, where appropriate, other beta-lactams against a total of 301 recent clinical isolates and characterized beta-lactamase producers. An agar dilution procedure was used to determine MICs and two inocula (10(4) and 10(6) cfu) were used throughout. MICs of the combination of ticarcillin and clavulanic acid were expressed as MICs of ticarcillin in the presence of 5 or 10 mg/l of clavulanic acid. Against most members of the Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii and Serratia marcescens), ticarcillin plus clavulanic acid was as active as or more active than piperacillin. Against Staphylococcus aureus. Haemophilus influenzae and the Bacteroides fragilis group the combination was considerably more active than piperacillin. Piperacillin was more active than the ticarcillin/clavulanic acid combination against Pseudomonas aeruginosa.