Tolerance of staphylococcus aureus to ß-lactam antibiotics

Penicillin has a bactericidal action on actively dividing bacteria. If protein synthesis of bacteria exposed to penicillin is inhibited, for example by the addition of chloramphenicol or the omission of an essential amino acid from the medium, the bactericidal action of penicillin changes into bacteriostasis (6). Rogers (10) and Shockman (11) found with S. aureus and Streptococcus faecalis respectively that inhibition of protein synthesis by chloramphenicol was accompanied by a sharp decline in the autolytic activity of the cells. These results supported their hypothesis that bacteria are lysed under the influence of penicillin because the equilibrium between peptidoglycan synthesis and autolysis shifts in the direction of the latter process as a result of the action of the ß-lactam antibiotic. The correctness of the hypothesis of Rogers and Shockman was supported by the work of Tomasz et al. (13), who isolated an autolysin-deficient mutant of Streptococcus pneumoniae which was not killed after exposure to penicillin. Tomasz et al. (13) called the absence of the bactericidal action of penicillin, tolerance for this agent. Since then, tolerant strains belonging to various species of bacteria have been isolated from clinical material. In previous studies (3,4) we showed that S. aureus strains can be divided into tolerant and nontolerant strains on the basis of their survival in vitro in the presence of high concentrations of methicillin ( >:- 64 .ug/ml). According to the observations of Rogers (10), the mechanism of the reduced lysis in tolerant S. aureus strains exposed to high concentrations of methicillin could be based on inhibition of protein synthesis. The existence of such a link has already been demonstrated by Mychajlonka et al. (8) for tolerant Streptococcus mutans strains. In the present study we investigated peptidoglycan, RNA and protein synthesis in two tolerant and two nontolerant S. aureus strains after exposure to a low and a high concentration of methicillin

Improved performance of PACE 2 with modified collection system in combination with probe competition assay for detecti

The Gen-Probe PACE 2 assay (GP) in combination with a modified collection system was compared with cell culture (CC) for the detection of Chlamydia trachomatis in urethral specimens from males. Analysis of discordant results was performed by PCR. The modifications, i.e., application of a more rigid swab type and a 50% reduction in the amount of transport medium, were made to improve the sensitivity of the assay. By using the modified GP on 302 urethral specimens from males, a sensitivity of 89.5% and a specificity of 100% were determined. In addition, performance of a probe competition assay on all GP samples with a result > 0.6 and < 1.0 times the cutoff factor (gray zone) detected three more true-positive samples. The sensitivity of GP in combination with the probe competition assay increased to 94.9%, with a specificity of 100%. This was identical to the performance of CC. The modified GP offers a very sensitive and specific alternative to CC

Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory

Comparative study of the effects of ceftizoxime, piperacillin, and piperacillin-tazobactam concentrations on antibacterial activity and selection of antibiotic-resistant mutants of Enterobacter cloacae and Bacteroides fragilis in vitro and in vivo in mixed-infection abscesses

The effects of ceftizoxime (CZX), piperacillin (PIP), and PIP-tazobactam (PT) concentrations on the antibacterial activity and selection of resistant mutants of Bacteroides fragilis and Enterobacter cloacae were investigated in vitro in a mixed-culture anaerobic time-kill study and in vivo in a mixed-infection abscess model. Mixed cultures were incubated for 24 h with 0.125 to 512 micro g of CZX per ml or 0.125 to 2,048 micro g of PIP or PT per ml. Mice were treated every 2 h for 24 h with CZX at 6 to 1,536 mg/kg/day or with PIP or PT at 24 to 6,144 mg/kg/day starting 30 min before inoculation with different B. fragilis-E. cloacae combinations. There was a good correlation between the in vitro and in vivo activities of the antibiotics and their MICs obtained with high inocula (10(8) CFU/ml). The respective 50% effective doses (milligrams per kilogram per day) with B. fragilis and E. cloacae 22491 were 771 and 521 for CZX, 416 and 643 for PIP, and 85 and 554 for PT, and with the B. fragilis-E. cloacae 032349 combination, they were 81 and 21 for CZX and 77 and 766 for PT. Resistant mutants of E. cloacae 22491 were preferentially selected in vitro with 2 to 64 micro g of CZX per ml and in vivo with CZX at 12 to 384 mg/kg/day. There was no preferential selection of CZX-resistant B. fragilis or E. cloacae 032349. For CZX-resistant E. cloacae 22491, we found a 16- to 512-fold increase in the MIC of CZX and increased MICs of other expanded-spectrum cephalosporins, owing in part to the production of a stably derepressed cephalosporinase. In vitro and in vivo, PT did not select resistant mutants of E. cloacae and B. fragilis. Results demonstrate the adverse microbiological outcome of choosing an expanded-spectrum cephalosporin like CZX for empirical treatment of mixed infections involving a susceptible Enterobacter strain

The accuracy of four commercial broth microdilution tests in the determination of the minimum inhibitory concentration of colistin

Colistin is considered as one of the last-resort antibiotics and reliable antimicrobial susceptibility testing is therefore crucial. The reference standard for AST according to EUCAST and CLSI is broth microdilution (BMD). However, BMD is labor intensive to perform. Commercial antimicrobial susceptibility tests derived from BMD method are available. We investigated the performance of four different commercial tests: Sensititre™, SensiTest™ Colistin, Micronaut MIC Strip Colistin and UMIC Colistin using 70 clinical isolates (half of them was deemed by VITEK2 as resistant), including isolates from cystic fibrosis patients and mcr-1 bearing isolates. We used two reference standards: BMD and composite MIC as determined by all four tests. Sensititre™ had essential agreement (EA, defined as minimum inhibitory concentration within ± 1 dilution) of 87% and 89% compared to BMD and composite reference standard, respectively. For SensiTest™, the EA’s were 93% and 90%. For UMIC, 87% and 90%, and for Micronaut, 83% and 84%. All four tests demonstrated categorical agreement (CA) above 90%. CA for SensiTest™ and Micronaut was both 96%, UMIC 94%, and Sensititre™ 93%. All tests were reproducible as tested in two quality control isolates. In conclusion, in clinical isolates from a large referral center, the four commercial tests for determination of colistin minimum inhibitory concentrations showed acceptable performance

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics

Molecular typing of Salmonella typhi strains from Dhaka (Bangladesh) and development of DNA probes identifying plasmid-encoded multidrug-resistant isolates

Seventy-eight Salmonella typhi strains isolated in 1994 and 1995 from patients living in Dhaka, Bangladesh, were subjected to phage typing, ribotyping, IS200 fingerprinting, and PCR fingerprinting. The collection displayed a high degree of genetic homogeneity, because restricted numbers of phage types and DNA fingerprints were observed. A significant number of the S. typhi strains (67%) were demonstrated to be multiple drug resistant (MDR). The vast majority of the MDR strains were resistant to chloramphenicol, ampicillin, trimethoprim, streptomycin, sulfamethoxazole, and tetracycline (R type CATmSSuT), a resistance phenotype that has also frequently been observed in India. Only two strains displayed a distinct MDR phenotype, R type AT-mSSuT. Pulsed-field gel electrophoresis demonstrated the presence of large plasmids exclusively in the MDR strains of both R types. The plasmids present in the S. typhi strains of R type CATmSSuT could be conjugated to Escherichia coli and resulted in the complete transfer of the MDR phenotype. PCR fingerprinting allowed discrimination of MDR and susceptible strains. The DNA fragments enabling discrimination of MDR and susceptible S. typhi strains by PCR were useful genetic markers for identifying MDR encoded by large plasmids of the H1 incompatibility group

PCR monitoring of response to liposomal amphotericin B treatment of systemic candidiasis in neutropenic mice

When a diagnosis of invasive candidiasis has been made, treatment with toxic fungicidal agents is inevitable. The crucial decision of when to stop such treatment is difficult to make, because cultures are often negative despite ongoing invasive candidiasis and can therefore not be used as a reliable parameter of effective therapy. In the present study, the use of PCR in monitoring the therapeutic efficacy of antifungal treatment with liposomal amphotericin B was evaluated by using neutropenic mice with systemic candidiasis. Blood cultures of infected mice treated with different doses of liposomal amphotericin B were only positive at the early onset of the infection process and became sterile within 3 days; this was true even with mice treated with 1 mg of liposomal amphotericin B per kg of body weight that experienced a relapse of infection 14 days later. A significant correlation between presence of Candida albicans in the kidneys and PCR results obtained with blood was demonstrated. Thus, PCR results obtained with blood samples correlated well with the therapeutic efficacy of antifungal treatment

Amoxicillin pharmacokinetics in preterm infants with gestational ages of less than 32 weeks

The multiple-dose pharmacokinetics of amoxicillin (AM [administered twice daily in a 25-mg/kg of body weight intravenous dose]) in 17 preterm infants (11 males; gestational age, 29 +/- 1.9 weeks; birth weight, 1,175 +/- 278 g) were evaluated on day 3 of life. Blood samples were collected from an arterial catheter at 0, 0.5, 1, 2, 4, 8, and 12 h after the intravenous dose. A high-performance liquid chromatography method was used to determine AM concentrations in serum. AM pharmacokinetics followed a one-compartment open model. The glomerular filtration rates of all patients were simultaneously studied by means of the 24-h continuous inulin infusion technique. The elimination half-life, apparent volume of distribution, and total body clearance of AM (mean +/- standard deviation) were 6.7 +/- 1.7 h, 584 +/- 173 ml, and 62.4 +/- 23.3 ml/h, respectively. The mean (+/- standard deviation) AM peak and trough levels were 53.6 +/- 9.1 and 16.0 +/- 4.9 mg/liter, respectively. All infants had a serum trough level above 5 mg/liter. The total body clearance and apparent volume of distribution of AM and the clearance of inulin increased significantly with increasing gestational age. The total bod

Improved detection of Candida albicans by PCR in blood of neutropenic mice with systemic candidiasis

A PCR using primers aimed at the multicopy gene coding for the small subunit rRNA and resulting in the synthesis of a 180-bp fragment was evaluated for its use in diagnosing invasive candidiasis in comparison with blood culture. With the use of a C. albicans-specific probe, +/- 10 to 15 C. albicans cells are detected in 100 microliters of whole blood by Southern analysis. A DNase pretreatment was critical in the purification process of yeast DNA from whole blood. Omission of the DNase pretreatment decreased assay sensitivity 10-fold. PCR analysis of blood specimens collected from mice with invasive candidiasis is more sensitive than blood culture (100 versus 67%, respectively) at 72 h after intravenous (i.v.) inoculation with C. albicans. Furthermore, the intensity of the hybridization signals increased with the progression of infection. In contrast, multiple blood samples from gastrointestinally colonized mice were all negative by PCR, indicating that the PCR assay is also specific and may, therefore, make a positive contribution to the detection and follow-up of invasive candidiasis