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PCR identification and automated ribotyping of Pseudomonas aeruginosa clinical isolates from intensive care patients.

Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant. P. aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period. Antimicrobial susceptibility of the 56 P. aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p < 0.01). In addition, 55% of the P. aeruginosa clinical isolates were resistant to 4 or more antibiotics. Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p < 0.05). PCR (using P. aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P. aeruginosa isolates tested. Automated ribotyping was used to investigate the clonal diversity of the 56 P. aeruginosa isolates. Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one. The P. aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time. The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P. aeruginosa infections.

Anti-Bacterial Agents↗

Relationship of antibiotic resistance phenotype to the R-pyocin susceptibility pattern in clinical isolates of Pseudomonas aeruginosa.

One hundred sixteen clinical isolates of Pseudomonas aeruginosa were collected from 7 hospitals in Athens. All strains were studied for their susceptibility to cefotaxime, ceftazidime, carbenicillin, aztreonam, imipenem, nalidixic acid, ciprofloxacin, gentamicin, and chloramphenicol. In addition, the R-pyocin susceptibility pattern was determined and the strains were O-serotyped and tested for their agglutination in acriflavine. The isolates included 53 strains resistant to both gentamicin and carbenicillin, 13 to carbenicillin only, 20 to gentamicin only, and 30 sensitive to gentamicin and carbenicillin. The multiresistant isolates displayed relatively higher resistance to all other antibiotics except aztreonam and cefotaxime. Remarkably 30 out of 53 multiresistant isolates reacted with one pyocin only, namely pyocin R2. This R-pyocin response was not encountered in any other strains of the other antibiotic resistance phenotypes. These isolates belonged to the 0-12 serogroup. The 0-12 serogroup was represented only in a minority of strains giving other R-pyocin reactions. It is interesting that strains reacting with pyocin R5 only were mostly susceptible to antibiotics. The results clearly indicate lipopolysaccharide-core mutations in multiresistant clinical isolates of P. aeruginosa. Despite the fact that the R-pyocin resistance pattern can not define the precise possible defect, the multiple and high level resistance associated with R2-pyocin reaction seems to be an interesting trait.

Bacteriocins↗

Epidemiology of antibiotic resistance in human isolates of Enterobacteriaceae in Sicily.

The authors have studied the antimicrobial susceptibility of 1073 clinical isolates of various genera of Enterobacteriaceae (collected during the period July-December 1988) to ampicillin, piperacillin, cefotaxime, ceftazidime, ceftriaxone, aztreonam, imipenem, gentamicin, amikacin, netilmicin, norfloxacin, and ciprofloxacin. Antimicrobial susceptibility was determined by Bauer-Kirby disk diffusion method. Of 1073 tested bacteria, 704 (65.6%) produced beta-lactamase detectable by nitrocefin test. The highest percentage of resistant strains occurred with ampicillin (70%) followed by piperacillin (24%) and cefotaxime (19%). Lower percentages of resistant strains were found for gentamicin (10%), aztreonam (8%), netilmicin (7%), norfloxacin (5%) and amikacin (4%). Two percent of the strains were resistant to ciprofloxacin and 0.5% to imipenem. The incidence of resistance in Klebsiella sp., Enterobacter sp., E. coli and Proteus sp. was compared to that found among 872 strains isolated during July-Dec. 1984. In all the Enterobacteriaceae, mainly Enterobacter sp., the increase in the resistance was high for ampicillin, piperacillin and cefotaxime and lower for gentamicin.

Drug Resistance, Microbial↗

In-vitro susceptibility of clinical isolates of Serratia species to antimicrobial agents.

The in-vitro activities of 12 antimicrobial agents against a total of 80 clinical isolates of Serratia marcescens and Serratia liquefaciens were determined by a broth microdilution method. Ampicillin and cefazolin were totally inactive against these organisms. The other beta-lactam antibiotics such as piperacillin, cefotaxime, ceftazidime, and the aminoglycosides such as gentamicin, tobramycin and netilmicin showed poor or moderate activity against Serratia isolates. Aztreonam and amikacin inhibited most of the strains tested. Imipenem and ciprofloxacin were very active in inhibiting all strains. Within the genus, S. liquefaciens was more resistant to aztreonam, ceftazidime and amikacin than S. marcescens.

Aminoglycosides↗

In-vitro activity of meropenem, a new carbapenem, against imipenem-resistant Pseudomonas aeruginosa and Xanthomonas maltophilia.

The activity of meropenem, a new carbapenem, as well as imipenem, ceftazidime, aztreonam, tobramycin, amikacin and ciprofloxacin against 18 strains of Xanthomonas maltophilia and 23 strains of Pseudomonas aeruginosa resistant to imipenem was tested. All strains of X. maltophilia were resistant to both penems. Ceftazidime, tobramycin and ciprofloxacin were the most active antimicrobial agents against this specie. 17% of imipenem-resistant strains of P. aeruginosa were sensitive to meropenem. Ciprofloxacin, amikacin and aztreonam were the most effective agents against these strains.

Anti-Bacterial Agents↗

In-vitro activity of ciprofloxacin and sixteen other antimicrobial agents against blood culture isolates.

The in-vitro antibacterial activities of seventeen antimicrobial agents including ampicillin, amikacin, Augmentin, aztreonam, cefazolin, cefuroxime, cefotaxime, ceftizoxime, ceftriaxone, ciprofloxacin, cloxacillin, erythromycin, gentamicin, penicillin G, piperacillin and vancomycin were compared against 100 Gram-negative and Gram-positive strains isolated from blood culture specimens received at the King Abdulaziz University Hospital (KAUH), in Jeddah, Saudi Arabia. The antibacterial susceptibility was determined by the minimal inhibitory concentration (MIC), using an agar dilution method. Ciprofloxacin exhibited the greatest activity, inhibiting 90% of the tested strains (MIC90) at a concentration ranging from < 0.015-0.5 mg/L. Against cloxacillin resistant or susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci, ciprofloxacin had similar activity with MIC90 of 0.2 mg/L. Salmonella typhi and salmonella species which were resistant to ampicillin and augmentin remained sensitive to ciprofloxacin (MIC90 < 0.015-0.125) mg/L.). Against gentamicin sensitive and resistant Pseudomonas aeruginosa and Pseudomonas species, ciprofloxacin MIC90 was 0.5 and 1 mg/L respectively. Aminoglycosides, third generation cephalosporins, aztreonam and antipseudomonal penicillins, on the other hand, showed high MIC90 well above the obtainable serum concentrations against Enterobacteriaceae and Pseudomonas species.

Anti-Bacterial Agents↗

PER-1 extended-spectrum beta-lactamase production in an Alcaligenes faecalis clinical isolate resistant to expanded-spectrum cephalosporins and monobactams from a hospital in Northern Italy.

An Alicaligenes faecalis (FL-424/98) resistant to expanded-spectrum cephalosporins and aztreonam was isolated from the urine of an inpatient at the Intensive Care Unit of the Varese Hospital (Northern Italy) after antimicrobial chemotherapy with cefazolin, vancomycin, and amikacin. Clavulanic acid restored the activity of expanded-spectrum cephalosporins, suggesting the production of an extended-spectrum beta-lactamase (ESbetaL). A crude extract of FL-424/98 showed the presence of two beta-lactamase activities focusing at pH 5.3 and 7.6, respectively. The ESbetaL activity, purified by means of three chromatographic steps, was found to correspond to the pI 5.3 enzyme. Determination of kinetic parameters confirmed that the enzyme efficiently hydrolyzed expanded-spectrum cephalosporins and aztreonam. A colony-blot hybridization revealed the presence of blaPER-related sequences in FL-424/98, and sequencing confirmed the identity of this determinant with blaPER-1, previously detected in Pseudomonas aeruginosa, Acinetobacter, and Salmonella clinical isolates from Turkey. Finding of blaPER-1 in a species that can be part of the resident human microbiota raises the possibility that it could be an efficient shuttle for spreading of this resistance gene among other opportunistic pathogens that are normally members of the resident microbiota. Kinetic parameters determined for the PER-1 enzyme with some cephalosporin substrates were somewhat different from those previously reported.

Adult↗

Pharmacokinetics of systemically administered antibiotics in patients with thermal injury.

Patients who sustain thermal injury may require adjustments of antibiotic dosing because of pharmacokinetic alterations and pathological changes that occur in this patient subset. In general, studies of gentamicin, tobramycin, and amikacin have demonstrated lower elimination half-lives in burn patients than in nonburn patients and healthy volunteers. Other studies have shown a strong correlation between vancomycin clearance and creatinine clearance and the need for higher vancomycin dosages for burn patients than for nonburn patients. Studies of ceftazidime, ticarcillin, enoxacin, and aztreonam have shown increases in the volume of distribution or decreases in the maximum concentration achieved. Total and renal drug clearance of aztreonam was highly correlated with creatinine clearance. With imipenem, the half-life, clearance, and volume of distribution observed in burn patients were not significantly different from those in nonburn patients, although substantial interpatient variability existed. Imipenem clearance was significantly correlated with creatinine clearance. Individualization of antibiotic therapy for burn patients is recommended with use of measured serum concentrations of the antibiotic or creatinine clearance as an estimate of renal function.

Aminoglycosides↗

Factors that influence the evolution of beta-lactam resistance in beta-lactamase-inducible strains of Enterobacter cloacae and Pseudomonas aeruginosa.

Induction ratios were determined for beta-lactamase-inducible strains of Enterobacter cloacae and Pseudomonas aeruginosa by using 10 beta-lactam agents. For E. cloacae, pre-incubation with ceftriaxone, cefoxitin, cefamandole, cefoperazone, or imipenem produced significantly larger amounts of beta-lactamase than did pre-incubation with moxalactam, clavulanate, ceftazidime, or aztreonam. For P. aeruginosa, imipenem was the best inducer, whereas ceftriaxone, piperacillin, cefoperazone, cefamandole, clavulanate, and aztreonam were poor beta-lactamase inducers. The rate of emergence of resistance by E. cloacae p99 and P. aeruginosa ATCC 27853 did not correlate with the induction ratio of the selecting agent; however, a strong correlation was noted between the mutation rate and the ratio of the MIC to the concentration of selecting antibiotic used. Emergence of resistance is related to the MIC of the antibiotic and the concentration of antibiotic used to select for resistance and is independent of the efficacy of the beta-lactam inducer. Resistant mutants arise through both beta-lactamase-dependent and -independent mechanisms.

Anti-Bacterial Agents↗

Comparison between the in-vitro activity of new agents on Pseudomonas aeruginosa isolates from cystic fibrosis patients and other chronic infections.

The in-vitro activities of the newer antipseudomonal agents cefsulodin, ceftazidime, U-63196, aztreonam and piperacillin were compared, together with azlocillin, carbenicillin, ticarcillin and tobramycin against 98 strains of Pseudomonas aeruginosa. On all of the 43 isolates from cystic fibrosis patients and the 55 strains from other chronically infected patients, ceftazidime (MIC90: 5.0 and 2.8 mg/l respectively) and cefsulodin (MIC90: 8.2 and 3.1 mg/l) were the most active cephalosporins, and U-63196 the least active (MIC90: 40.1 and 46.1 mg/l). A high incidence of resistance (23 of 55 strains) to tobramycin was found only in the non-cystic fibrosis isolates. The activity of the other agents was comparable in the cystic fibrosis and non-cystic fibrosis isolates. MIC90 for aztreonam were 15.8 and 13.6 mg/l. Piperacillin was the most active penicillin in both populations (MIC90: 25.8 and 21.8 mg/l). Cross resistance was demonstrated in both populations for the penicillin compounds.

Anti-Bacterial Agents↗

In-vitro emergence of beta-lactam-resistant variants of Pseudomonas aeruginosa.

The frequency with which resistant variants could be found in Pseudomonas aeruginosa ATCC 27853 during growth in media with and without antibiotics was determined for ticarcillin, piperacillin, cefotaxime, latamoxef, cefoperazone, ceftriaxone, ceftazidime, aztreonam and imipenem. The resistance frequency was highest for ceftriaxone, cefotaxime, cefoperazone and piperacillin (up to 2 X 10(-4)) and lowest for ticarcillin, latamoxef, ceftazidime, aztreonam and imipenem (less than 10(-8)). The resistant variants showed cross-resistance to all of the beta-lactam antibiotics tested with the exception of imipenem. The MIC profiles of all variants were similar regardless of which antibiotic the cultures had been exposed to previously. Although all resistant variants produced and excreted cephalosporinases, increased resistance was demonstrated to both cephalosporinase-susceptible and cephalosporinase-stable beta-lactam antibiotics.

Anti-Bacterial Agents↗

Comparative in-vitro activity of tigemonam, a new monobactam.

The in-vitro activity against Gram-negative aerobic bacterial pathogens of a new oral monobactam, tigemonam, was compared with those of amoxycillin/clavulanic acid, aztreonam, cefaclor, ceftazidime, cefuroxime, ciprofloxacin, co-trimoxazole and gentamicin, by an agar-dilution method. Tigemonam inhibited 90% of strains tested of Escherichia coli, Klebsiella spp., Proteus spp., Salmonella spp., Haemophilus influenzae and Branhamella catarrhalis at concentrations of 0.25 mg/l or below. The MIC90 for Enterobacter spp. was 16 mg/l and for Citrobacter 4 mg/l. Pseudomonas aeruginosa was resistant to tigemonam but susceptible to aztreonam. In comparison with the other oral antibiotics tested, tigemonam was generally more active. Tigemonam showed minimal inoculum dependence, between 10(2) and 10(6) cfu per spot. It is concluded that tigemonam is a candidate for clinical trials in patients with infections caused by Gram-negative aerobes other than pseudomonas and acinetobacter.

Anti-Bacterial Agents↗

In-vitro activity of tigemonam, an oral monobactam, against gram-negative rods, including variants in beta-lactamase-production.

Tigemonam was compared in vitro with other orally active beta-lactams, aztreonam and ciprofloxacin against a selection of Gram-negative clinical isolates and laboratory-derived beta-lactamase-producing variants. Of the orally active beta-lactams, tigemonam was the most potent, with a spectrum of activity similar to that of aztreonam. This included stability to plasmid-mediated beta-lactamases and poor induction of chromosomal beta-lactamases. The susceptibility to Class I enzymes was only clinically significant for derepressed Enterobacter cloacae mutants. Tigemonam may have a valuable role in the management of infection caused by enterobacteria, particularly if bioavailability following oral administration is confirmed in human subjects.

Anti-Bacterial Agents↗

In-vitro activity of the penem, FCE 22101, and tentative interpretive criteria for disc susceptibility tests.

The in-vitro activities of two penems (FCE 22101 and SCH 34343) were compared to those of a carbapenem (imipenem), a monobactam (aztreonam) and three cephalosporins (cefoperazone, ceftazidime, and cefotaxime). FCE 22101 displayed a broad spectrum of antibacterial activity against both Gram-negative and Gram-positive pathogens, including anaerobic bacteria. Imipenem and aztreonam, however, were a little more potent against susceptible species. FCE 22101 was bactericidal against 14 of 18 isolates tested; one strain each of Enterobacter cloacae, Staphylococcus aureus (methicillin sensitive), Staph. aureus (methicillin resistant) and Streptococcus faecalis were not killed by FCE 22101. For disc diffusion tests, a 10 micrograms FCE 22101 disc is tentatively recommended with zones of less than or equal to 13 mm for resistant (MIC greater than 8.0 mg/l) and greater than or equal to 17 mm for susceptible (MIC less than or equal to 4.0 mg/l). Those criteria need to be reevaluated when additional pharmacokinetic information and clinical experience have been gathered.

Anti-Bacterial Agents↗

In-vitro activity of FCE 22101 and other beta-lactam antibiotics against Enterobacteriaceae resistant to third generation cephalosporins.

The in-vitro activity of FCE 22101 was compared with those of imipenem, aztreonam, cefotaxime, ceftriaxone and latamoxef against 225 Enterobacteriaceae (209 clinical isolates and 16 derivatives of Escherichia coli K12) chosen for their resistance (MIC greater than or equal to 8 mg/l) to third-generation cephalosporins. The beta-lactamases produced by the strains of Enterobacter cloacae, Klebsiella pneumoniae and Esch. coli were identified. The mean MICs of FCE 22101 were 2-8 mg/l for Ent. cloacae, Citrobacter freundii and Morganella morganii. For Ent. cloacae, the MICs were slightly higher for the cephalosporinase non overproducing (5.5-8 mg/l) than for the cephalosporinase overproducing (4.5 mg/l) strains. The mean MIC was 9 mg/l for Serratia spp. but for half of the strains the MICs were greater than or equal to 16 mg/l. The MICs of FCE 22101 were 0.5-1 mg/l for the resistant clinical isolates or isogenic derivatives of K. pneumoniae and Esch. coli producing extended broad-spectrum beta-lactamases (SHV-2, SHV-3 or CTX-1), whether measured in the presence of clavulanate or not, and against the sensitive controls. This showed that the compound was not affected by these new beta-lactamases. The MICs of imipenem, on the average four to five times lower than those of FCE 22101, were found to be very similar for resistant and sensitive strains. The MICs of aztreonam were high (32-128 mg/l) for C. freundii, K. oxytoca and cephalosporinase overproducing strains of Ent. cloacae. There was cross-resistance between the third-generation cephalosporins, but the MICs of latamoxef remained low (1 mg/l) against M. morganii, Klebsiella spp. and Esch. coli.

Carbapenems↗

Comparative study of five plasmid-mediated ceftazidimases isolated in Klebsiella pneumoniae.

Five plasmid-mediated beta-lactamases conferring a high level of resistance to ceftazidime were isolated from Klebsiella pneumoniae strains. These ceftazidimases (CAZ) differed in their isoelectric point (from 5.3 to 8.2) and were encoded by large self-transferable plasmids of 85 kb (CAZ-2, CAZ-3) or greater than or equal to 150 kb (CAZ-1, CAZ-4, CAZ-5). The 85 kb plasmids seemed closely related to pCFF04 encoding CTX-1 enzyme and belonged to the same incompatibility group 7 or M. These beta-lactamases hydrolysed all beta-lactams with the exception of cephamycins and carbapenems. For CAZ-1, CAZ-2 and CAZ-3 producers, MICs of ceftazidime (32-256 mg/l) were higher than MICs of cefotaxime (0.12-2 mg/l) and aztreonam (1-16 mg/l). For the strains producing the beta-lactamases CAZ-4 and CAZ-5, MICs of aztreonam were the highest (greater than or equal to 256 mg/l). The impaired activities of cephalosporins and monobactams were restored equally well by 2 mg/l of clavulanate, sulbactam and CL-298741 for CAZ-2 producing strains (wild type and transconjugant). Sulbactam (2 mg/l) had a lower protective effect than other inhibitors on ceftazidime for CAZ-1 and CAZ-3 producing K. pneumoniae. The protective effect of sulbactam (2 mg/l) was lower than that of the other inhibitors on all beta-lactams for CAZ-4 and CAZ-5 producers. The enzymes CAZ-1, CAZ-2 and CAZ-3 derived from TEM beta-lactamase whereas CAZ-4 and CAZ-5 derived from SHV-1 enzyme.

Autoradiography↗

Prevalence of a transferable SHV-5 type beta-lactamase in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Greece.

Analysis with a double-disc synergy test (DDST) of clinical isolates of Klebsiella pneumoniae and Escherichia coli during the period October 1988-September 1989 revealed that 24% of the former and 4% of the latter, mainly isolated from urine, possessed an extended-spectrum beta-lactamase. During this period no DDST was positive for isolates of other enterobacterial species. Transfer of ceftazidime resistance was demonstrated from six K. pneumoniae and two E. coli isolates resistant to third generation cephalosporins and aztreonam. Apart from one strain of K. pneumoniae, these strains harboured self-transferable multiresistance plasmids (c. 91 kb) with closely related EcoRI, HindIII, AvaII and PstI restriction patterns. These plasmids encoded an extended-spectrum beta-lactamase that conferred an unusually high level of resistance to ceftazidime and aztreonam (MIC greater than or equal to 64 mg/l). This enzyme had a pI of 8.2 and a substrate profile similar to that of the SHV-5 enzyme isolated initially in Chile, and later in France (CAZ-4). The remaining K. pneumoniae isolate harboured a transmissible multiresistance plasmid (c. 182 kb) that encoded the widely distributed SHV-2 enzyme. The resistance to cefoxitin that was observed in some of these strains was associated with outer membrane protein alterations.

Bacteria↗

Clinical isolates of Escherichia coli producing TRI beta-lactamases: novel TEM-enzymes conferring resistance to beta-lactamase inhibitors.

Two different strains of Escherichia coli exhibiting unusual patterns of resistance to beta-lactam antibiotics were isolated from patients at Cochin Hospital. Both isolates showed a low level of resistance to amoxycillin, ticarcillin and ureidopenicillins but were susceptible to cephalosporins, aztreonam and imipenem; beta-lactamase inhibitors potentiated the activities of the beta-lactams to only a limited extent. All resistance characteristics of the strains were transferable by conjugation to E. coli K12. Resistance was shown to be due to beta-lactamases of pI 5.20 and relative molecular masses of 24,000. The hydrolytic and inhibition profiles of these enzymes were similar to each other but differed from those of broad-spectrum beta-lactamases (TEM-1). The rates of hydrolysis (Vmax) of amoxycillin (c. 200%) were higher than that for TEM-1 (84%). Ticarcillin, ureidopenicillins and cephaloridine were hydrolyzed slowly. However, as for TEM-1, no hydrolysis was observed with cefoxitin, third generation cephalosporins, aztreonam and imipenem. The high Km values demonstrated the poor affinity of these enzymes for their substrates. Unlike TEM-1, they were poorly inhibited by beta-lactamase inhibitors. These two enzymes differed from each other as follows: (i) the concentrations of clavulanic acid required for 50% beta-lactamase inhibition were 31 mumol/L for one enzyme (E-SAL) and 9.4 mumol/L for the other (E-GUER); (ii) p-chloromercuribenzoate was a more active inhibitor of E-SAL then E-GUER. The titration curve method and DNA-DNA hybridization studies demonstrated that both enzymes were structurally related to TEM-1. The novel plasmid-encoded enzymes produced by the two isolates of E. coli appeared to be almost identical and to be derived from TEM-enzymes. On the basis of their presumed phylogeny and their biological properties, we propose that these beta-lactamases be given the generic name TRI (TEM Resistant to beta-lactamase Inhibitors).

Anti-Bacterial Agents↗