Clinical significance of cerebrospinal fluid inhibitory titers of antibiotics, based on pharmacokinetic/pharmacodynamic parameters, in the treatment of bacterial meningitis.

The cerebrospinal fluid (CSF) inhibitory titer (CSF-IT) of an antibiotic, which can be used to estimate the duration of time above the agent's MIC in the CSF, was introduced as one of the indices to evaluate the effectiveness of antibiotic selection in treating bacterial meningitis. The CSF-IT was determined via a microdilution method. A suspension of the causative organism was added to a tube containing twofold diluted CSF and double-concentrated Mueller-Hinton broth with supplement. The CSF-IT was determined by the maximum point without turbidity of medium after overnight incubation at 37 degrees C. Concerning the strain of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae (BLNAR), the killing rates of both meropenem and piperacillin were compared in an in vitro pharmacokinetic (PK) model, in which human pharmacokinetics in CSF were simulated. Organisms recovered from the CSF in 37 treated clinical cases of bacterial meningitis were H. influenzae, Streptococcus agalactiae, Streptococcus pneumoniae, Escherichia coli, and Neisseria meningitidis; in these cases, the CSF-IT ranged from 1: 8 to as high as 1: 4 096. In the in vitro PK model, the concentrations of both drugs were higher than the MICs over a period of 24 h; however, the killing rate of piperacillin was higher than that of meropenem, and bacterial regrowth was observed after the administration of meropenem. A CSF-IT value higher than 1: 32 indicates that the antibiotic concentration in the CSF exceeds the MIC for 24 h. The effect of piperacillin on BLNAR depends not only on the time above MIC of 24 h but also on the maximum concentration in the CSF.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli.

We developed a loop-mediated isothermal amplification (LAMP) assay for the rapid and simple detection of Campylobacter jejuni and Campylobacter coli. The assay provides a specific LAMP product for each of these two species. The assay correctly identified 65 C. jejuni and 45 C. coli strains, but not 75 non-C. jejuni/coli strains. The sensitivity of the LAMP assay for C. jejuni and C. coli in spiked human stool specimens was 5.6 x 10(3) c.f.u. g(-1) (1.4 c.f.u. per test tube) and 4.8 x 10(3) c.f.u. g(-1) (1.2 c.f.u. per test tube), respectively. When 90 stool specimens from patients with diarrhoea were tested by LAMP and direct plating, the LAMP results showed 81.3 % sensitivity and 96.6 % specificity compared to isolation of C. jejuni and C. coli by direct plating. Further, the LAMP assay required less than 2 h for detection of C. jejuni and C. coli in stool specimens. This LAMP assay is a rapid and simple tool for the detection of C. jejuni and C. coli and will be useful in facilitating the early diagnosis of food poisoning incidents caused by these organisms.

Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis.

A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

In vitro activities of piperacillin against beta-lactamase-negative ampicillin-resistant Haemophilus influenzae.

The in vitro activities of piperacillin (PIP) against beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae were compared with those of cefotaxime (CTX) and ceftriaxone (CRO), and the potency of PIP as therapy for meningitis caused by BLNAR is also discussed. PIP showed good activity (MIC at which 90% of strains are inhibited, 0.25 micro g/ml) against 69 BLNAR strains, and its activity was comparable to that of CRO and superior to that of CTX. No significant correlation was observed between the MICs of PIP and CTX or CRO or AMP, whereas a high correlation was observed between the MICs of CTX and CRO. In the killing study, PIP showed potent bactericidal activity compared with those of CTX and CRO. By microscopic examination, PIP caused the formation of a spindle and short filamentous cells with bulges and induced cell lysis in BLNAR strains, while treatment with CTX and CRO resulted in the formation of large, spherical cells without any obvious lysis. The affinity of Bocillin FL, a fluorescent penicillin used for determination of the 50% inhibitory concentration (IC(50)s) for penicillin-binding proteins (PBPs), to PBPs 3a and 3b of BLNAR strains was drastically decreased compared with that to an AMP-susceptible strain (ATCC 33391). In the case of the BLNAR strains, the IC(50)s for PBPs 1a, 1b, and 2 were similar to those for the PBPs of ATCC 33391. Since the affinity of binding to PBPs 3a and 3b of the BLNAR strains decreased drastically, the second targets among the PBPs were PBP 2 for PIP, PBP1 (1a and 1b) for CTX and CRO. In conclusion, PIP showed excellent activities against BLNAR strains in a manner different from those of cephem antibiotics, suggesting that it could be a candidate therapeutic agent for the treatment of meningitis caused by BLNAR strains.

Prevalence of cefotaxime resistance in group B streptococcus isolates from Osaka, Japan.

Streptococcus agalactiae (group B streptococcus; GBS) organisms are a major cause of severe infections, including bacteremia and meningitis in newborns. According to previous reports, GBS organisms are uniformly sensitive to penicillin G (PCG). The susceptibility of 117 strains isolated at Yodogawa Christian Hospital in Osaka, Japan, in 2001 was examined with the WalkAway system, using currently valid National Committee for Clinical Laboratory Standards (NCCLS) interpretive criteria. Twenty-one strains (18%) had intermediate susceptibility and 1 strain (1%) was resistant to PCG. Fifty-one strains (44%) had intermediate susceptibility to ampicillin (ABPC). No ABPC-resistant strain was found. Six GBS strains were selected from the 51 strains showing intermediate susceptibility to ABPC to determine the minimal inhibitory concentrations (MICs). The MICs of the 6 strains were: 1 microgram/ml to ABPC, 0.25 microgram/ml to PCG, 2 micrograms/ml to cefotaxime (CTX), 0.016 microgram/ml to panipenem (PAPM), and more than 4 micrograms/ml to erythromycin (EM). These 6 strains were distinctly resistant to CTX. Peak concentrations in excess of three to ten times the bactericidal concentrations at the site of infection are associated with the best clinical response. In meningitis caused by GBS whose susceptibility is intermediate or resistant to PCG or ABPC, it is difficult to maintain a sufficient therapeutic concentration in cerebrospinal fluid after the administration of these two agents. It is preferable to use PAPM, because the efficacy and safety of PAPM in the treatment of purulent meningitis caused by penicillin-resistant Streptococcus pneumoniae were established in Japan.