Epidemiological cut-off values for Flavobacterium psychrophilum MIC data generated by a standard test protocol.

Epidemiological cut-off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut-off values. For these agents, the cut-off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L(-1) for erythromycin, ≤2 mg L(-1) for florfenicol, ≤0.025 mg L(-1) for oxolinic acid (OXO), ≤0.125 mg L(-1) for oxytetracycline and ≤20 (1/19) mg L(-1) for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild-type observations were 'off scale', and therefore, statistically valid cut-off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut-off could not be determined. For flumequine, the putative wild-type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild-type and non-wild-type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10.

The MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification.

Effect of incubation temperature and time on the precision of data generated by antibiotic disc diffusion assays.

The influence on the precision of disc diffusion data of the conditions under which the tests were performed was examined by analysing multilaboratory data sets generated after incubation at 35 °C for 18 h, at 28 °C for 24 h and 22 °C for 24 h and 48 h. Analyses of these data sets demonstrated that precision was significantly and progressively decreased as the test temperature was reduced from 35 to 22 °C. Analysis of the data obtained at 22 °C also showed the precision was inversely related to the time of incubation. Temperature and time related decreases in precision were not related to differences in the mean zone sizes of the data sets obtained under these test conditions. Analysis of the zone data obtained at 28 and 22 °C as single laboratory sets demonstrated that reductions of incubation temperature resulted in significant increases in both intralaboratory and interlaboratory variation. Increases in incubation time at 22 °C were, however, associated with statistically significant increases in interlaboratory variation but not with any significant increase in intralaboratory variation. The significance of these observations for the establishment of the acceptable limits of precision of data sets that can be used for the setting of valid epidemiological cut-off values is discussed.

Invasive disease caused by Haemophilus influenzae in Sweden 1997-2009; evidence of increasing incidence and clinical burden of non-type b strains.

Introduction of a conjugated vaccine against encapsulated Haemophilus influenzae type b (Hib) has led to a dramatic reduction of invasive Hib disease. However, an increasing incidence of invasive disease by H. influenzae non-type b has recently been reported. Non-type b strains have been suggested to be opportunists in an invasive context, but information on clinical consequences and related medical conditions is scarce. In this retrospective study, all H. influenzae isolates (n = 410) from blood and cerebrospinal fluid in three metropolitan Swedish regions between 1997 and 2009 from a population of approximately 3 million individuals were identified. All available isolates were serotyped by PCR (n = 250). We observed a statistically significant increase in the incidence of invasive H. influenzae disease, ascribed to non-typeable H. influenzae (NTHi) and encapsulated strains type f (Hif) in mainly individuals >60 years of age. The medical reports from a subset of 136 cases of invasive Haemophilus disease revealed that 48% of invasive NTHi cases and 59% of invasive Hif cases, respectively, met the criteria of severe sepsis or septic shock according to the ACCP/SCCM classification of sepsis grading. One-fifth of invasive NTHi cases and more than one-third of invasive Hif cases were admitted to intensive care units. Only 37% of patients with invasive non-type b disease had evidence of immunocompromise, of which conditions related to impaired humoral immunity was the most common. The clinical burden of invasive non-type b H. influenzae disease, measured as days of hospitalization/100 000 individuals at risk and year, increased significantly throughout the study period.

Socioeconomic factors and antibiotic use in relation to antimicrobial resistance in the Amazonian area of Peru.

Our objective was to correlate antibiotic resistance in gut E. coli flora of children, aged 6-72 months, with use of antibiotics, socioeconomic status (SES) and household characteristics in the urban communities of Yurimaguas and Moyobamba in the Amazonian area of Peru. Caregivers of 1598 children were interviewed using a structured questionnaire in a cross-sectional survey. Faecal samples were collected from the children and the antimicrobial susceptibility of E. coli was analysed by a rapid resistance screening method. Significantly higher odds for resistance were seen for children who had used antibiotics, both during the last 2 weeks and the last 6 months. Children from wealthier families had significantly higher odds for resistance to a number of antibiotics than children from the least wealthy families (Yurimaguas: nalidixic acid, OR = 2.13; ciprofloxacin, OR = 2.09; chloramphenicol, OR = 1.98. Moyobamba: nalidixic acid, OR = 1.59; ciprofloxacin, OR = 1.69). Thus, the children of wealthier families had a significantly increased odds ratio for resistance, also when controlling for the family's antibiotic use. Unknown factors related to socioeconomic status seem to contribute to the results seen in the study area.

Two new dfr genes in trimethoprim-resistant integron-negative Escherichia coli isolates.

Two Escherichia coli isolates resistant to trimethoprim but negative for integrons carried two new resistance genes, dfrA24 and dfrA26, remotely similar to one another and to the cassette-independent genes dfrA8 and dfrA9. The dfrA24 gene was not associated with known mobile elements, while dfrA26 was associated with the CR1 common region.

Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values.

MIC distribution data were obtained from a variety of international sources, and pooled after selection by a defined criterion. Sixty-seven of these datasets were subjected to a range of statistical goodness-of-fit tests. The log-normal distribution was selected for subsequent modelling. Cumulative counts of MIC distribution data were fitted to the cumulative log-normal distribution using non-linear least squares regression for a range of data subsets from each antibiotic-bacterium combination. Estimated parameters in the regression were the number of isolates in the subset, and (the log(2) values of) the mean and standard deviation. Optimum fits for the cumulative log-normal curve were then used to determine the wild-type MIC range, determined by calculating the MICs associated with the lower and upper 0.1% of the distribution, rounding to the nearest two-fold dilution, and calculating the probabilities of values higher and lower than these values. When plotted logarithmically, histograms of MIC frequencies appeared normal (Gaussian), but standard goodness-of-fit tests showed that the two-fold dilution grouping of MICs fits poorly to a log-normal distribution, whereas non-linear regression gave good fits to population (histogram) log-normal distributions of log(2) MIC frequencies, and even better fits to log-normal cumulative distributions. Optimum fits were found when the difference between the estimated and true number of isolates in the fitted subset was minimal. Sixteen antibiotic-bacterium datasets were fitted using this technique, and the log(2) values of the means and standard deviations were used to determine the 0.1% and 99.9% wild-type cut-off values. When rounded to the nearest two-fold dilution, > or = 98.5% of MIC values fall within the cut-off value range. Non-linear regression fitting to a cumulative log-normal distribution is a novel and effective method for modelling MIC distributions and quantifying wild-type MIC ranges.

Meropenem susceptibility breakpoint for Pseudomonas aeruginosa strains hyperproducing mexB mRNA.

Twenty-five isolates of Pseudomonas aeruginosa with different meropenem susceptibilities were subjected to quantitative RT-PCR for analysis of transcription levels of oprD, mexB and mexD, and, in selected isolates, PA3720, which is hyper-expressed in nalC efflux mutants. Regulator genes of efflux pump MexAB-OprM, mexR and PA3721 (putative) were sequenced in selected isolates. The potential for mathematical reconstruction of the ideal susceptible population using normalised resistance interpretation (NRI) was also studied. In three isolates with intermediate susceptibility to meropenem (according to Swedish breakpoints), a reduction in MIC from 4 to 2 mg/L was observed with efflux inhibitor MC-207,110. These isolates would be considered susceptible according to British Society for Antimicrobial Chemotherapy and NCCLS breakpoints. These three isolates had between 4.6- and 5.0-fold increases in mexB transcription. None of these isolates had significant nalB mutations, but an Ala145-->Val mutation was observed in PA3721 in two of the isolates. However, these isolates had moderately increased production of PA3720 only. Single-strain regression analysis did not detect any major biological differences between the different groups. Using NRI, a disk-diffusion susceptibility breakpoint of >/= 28 mm was generated. Isolates with intermediate susceptibility to meropenem, which are considered fully susceptible in many countries, displayed possible low-grade meropenem resistance mechanisms, implying that the susceptibility breakpoint should be reconsidered. The increased transcription of mexB mRNA in such isolates seems unrelated to nalB or nalC mutations.

Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria.

Despite a trend of declining consumption, resistance to co-trimoxazole has increased during a 12-year period in Stockholm. The molecular background to this surprising development was investigated by using PCR to screen for integrons and specific resistance genes, followed by sequence analysis of selected integrons, in 105 clinical urinary isolates of Gram-negative bacteria selected partly for trimethoprim resistance. Sixty-five integrons of class 1 or 2 were detected in a subset of 59 isolates, and of these positive isolates, all but one were resistant to trimethoprim. However, 11 isolates were resistant to trimethoprim, but negative for integrons. Isolates positive for integrons were resistant to an average of 4.2 antibiotics, compared with 1.9 antibiotics for integron-negative isolates. Despite this, the only gene cassettes identified in 19 class 1 integrons analysed were dfr and aadA cassettes. Thus, only resistance to trimethoprim, streptomycin, spectinomycin and sulphonamides could be explained by the presence of integrons in these isolates. A new dfr gene, named dfrA22, was discovered as a single gene cassette in a class 1 integron. In addition, sulphonamide resistance in many isolates was caused by carriage of sul2, which has no known association with integrons. Resistance to co-trimoxazole and many other antibiotics was thus not accounted for fully by the presence of integrons in these isolates.

Effect of clonal and serotype-specific properties on the invasive capacity of Streptococcus pneumoniae.

The present study compares the molecular epidemiology of Streptococcus pneumoniae causing invasive disease and carriage, respectively, in one geographic area (Stockholm, Sweden) during a specific point in time (the year 1997). A total of 273 invasive isolates (257 from adults and 16 from children) obtained from the 2 major hospitals in Stockholm, as well as 246 nasopharyngeal isolates recovered from children attending 16 day-care centers in the Stockholm area, were analyzed by serotyping, molecular typing (by pulsed-field gel electrophoresis and multilocus sequence typing), and antibiotic susceptibility testing. Of the 34 different serotypes plus nontypeable strains identified in the present study, 12 were never found among the 246 colonizing isolates, whereas only 3 were never found among the 273 invasive isolates. The isolates formed 2 major classes: 1 class that was found mainly among invasive isolates (type 1, 4, 7F, and 9V isolates) and was clonally highly related and 1 class that caused invasive disease but was also common in carriage (including type 6A, 6B, 14, and 19F isolates) and was genetically more diverse. Clones were found that belonged to the same serotype but had different abilities to cause invasive disease. Also, isolates belonging to the same clone were found, although they had different capsules because of serotype switch, and were found to have the same disease potential. Hence, properties associated with a particular clonal type, in addition to capsular serotype, are likely to be important for the potential of pneumococci to cause invasive disease.

Patterns of mutations in target genes in septicemia isolates of Escherichia coli and Klebsiella pneumoniae with resistance or reduced susceptibility to ciprofloxacin.

Thirty-two Escherichia coli and 21 Klebsiella pneumoniae septicemia isolates with varying degrees of resistance to ciprofloxacin were analyzed for the presence of point mutations within the quinolone-resistance target genes. The number of mutations observed in the resistant isolates agreed with the level of ciprofloxacin resistance in both species. Such isolates were also resistant to nalidixic acid. Isolates with borderline susceptibility to ciprofloxacin, on the other hand, behaved differently in the two species. In E. coli all the isolates harbored at least one mutation and these isolates were also resistant to nalidixic acid, while no mutations were detected in the K. pneumoniae isolates, and susceptibility to nalidixic acid was unpredictable. Therefore, nalidixic acid cannot be used as a class representative. Time-kill curve studies on an isolate with borderline susceptibility from each species showed higher degrees of resistance to ciprofloxacin in comparison to that of the wild-type E. coli. A previously unreported parC mutation, S57-->T, was detected in a resistant E. coli isolate and might expand the QRDR of this gene. Normalized resistance interpretations of histograms confirmed the setting of microbiological zone breakpoints for ciprofloxacin testing.

Different trends in antibiotic resistance rates at a university teaching hospital.

OBJECTIVES: To investigate long-term trends in antibiotic resistance of common bacterial species isolated at a university hospital and in its intensive care units (ICUs). METHODS: Levels of antibiotic resistance of common bacterial pathogens were investigated at the Karolinska Hospital during the 12-year period 1988-99. Resistance rates were analyzed for the entire hospital, as well as for ICUs combined. RESULTS: At the Karolinska Hospital, we found increased ciprofloxacin resistance among Escherichia coli isolates, from 0% in 1991 to 11% in 1999. In the ICUs, the corresponding increase was from 0% to 4.8% during the same period. Co-trimoxazole resistance levels increased from 7.5% to 14%, with lower levels for the ICUs. For ampicillin, cefuroxime, and gentamicin, the levels of resistance were similar in the whole hospital and in the ICUs. Among Pseudomonas aeruginosa isolates, imipenem resistance was higher in the ICUs. For ciprofloxacin, resistance increased from 2.5% in 1991 to 13% in 1999 in the whole hospital, with similar figures for the ICUs. CONCLUSION: The resistance rates at the Karolinska Hospital were still generally low, but were increasing for some antibiotic-microbe combinations. The results emphasize the importance of including all sectors of a hospital in resistance surveillance studies, and also the value of long surveillance periods.

A new method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes.

OBJECTIVE: To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. METHODS: The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. RESULTS: The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. CONCLUSIONS: The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test.

Fluconazole and voriconazole multidisk testing of Candida species for disk test calibration and MIC estimation.

Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of susceptibility to fluconazole of < or =8 microg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-microg fluconazole disk was calculated to be > or =18 mm for C. albicans and > or =22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-microg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 microg/ml, whereas a 2.5-microg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.

Antibiotic medication and bacterial resistance to antibiotics: a survey of children in a Vietnamese community.

OBJECTIVE: To investigate antibiotic use and antibiotic susceptibility of respiratory tract pathogens in children aged 1-5 years in Bavi, Vietnam. METHOD: Nasopharynx and throat specimens were collected from 200 children from randomly selected households in a demographically defined population. Respiratory isolates were tested for antibiotic susceptibility according to the standard disk diffusion method. A questionnaire survey of carers elicited information on type of antibiotic used, duration of treatment, where the antibiotics had been purchased, type of treatment information retained by carers and episodes of illness preceding the study. RESULTS: 82% of the children had at least one symptom of acute respiratory tract infection (ARI) in the 4 weeks prior to the study, and of these 91% were treated with antibiotics. The most commonly used antibiotics were ampicillin (74%), penicillin (12%), amoxicillin (11%), erythromycin (5%), tetracycline (4%) and streptomycin (2%). Ampicillin was used for 3.3 days on average (SD:1.8) and penicillin for 2.6 days (SD:0.7). When deciding which antibiotic to use, 67% of the carers consulted the pharmacy seller, 11% decided themselves and 22% followed the doctor's prescription. The carrier rate of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis was 50%, 39% and 17%, respectively. Isolates from 145 children were susceptibility tested, and 74% were found to carry resistant pathogens. Of the tested isolates, 90% of S. pneumoniae, 68% of H. influenzae and 74% of M. catarrhalis were resistant to at least one antibiotic. The mean number of antibiotics (susceptible strains excluded) to which resistance was found was 2.0 (SD:1.2), 2.5 (SD:1.8) and 2.1 (SD:0.9), respectively. S. pneumoniae and H. influenzae showed high resistance to tetracycline (88% and 32%, respectively), trimethoprim/sulphonamide (32% and 44%), and chloramphenicol (25% and 24%). 23% of S. pneumoniae were erythromycin-resistant and 18% of H. influenzae isolates were resistant to ampicillin. There was a significant difference in ampicillin and penicillin resistance between the group of children previously treated with beta lactam antibiotics and the group of children who did not receive antibiotics. CONCLUSION: As reported by the carers, children in Bavi are treated with antibiotics frequently. Most antibiotics were obtained without consulting a doctor. High levels of antibiotic resistance and high prevalence of multidrug-resistant strains were found among respiratory pathogens. The existence of a large reservoir of resistance genes among children in low-income countries represents a threat to the success of antibiotic therapy throughout the world. Multi-faceted programmes to improve rational use of antibiotics in Vietnam are urgently needed.

Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex.

A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.

Fusidic acid disk diffusion testing of clostridium difficile can be calibrated using single-strain regression analysis.

Single-strain regression analysis (SRA) was employed to calibrate the disk diffusion antibiotic susceptibility test for fusidic acid and Clostridium difficile. MIC determinations of 40 clinical isolates of C. difficile were performed with the E-test. The disk diffusion test was standardized according to the Swedish Reference Group for Antibiotics (SRGA). Disks used for SRA contained 1.5, 5, 15, 50 and 150 microg fusidic acid and the routine disk contained 50 microg fusidic acid. A control strain, ATCC 9689, was also tested. SRA constants A and B of the regression lines were calculated. This permitted the determination of zone breakpoints for C. difficile. When applying the pharmacological MIC S and R limits set by SRGA to the E-test results I strain of C. difficile was interpreted as resistant. Zone breakpoints corresponding to the pharmacological MIC limits and calculated using the mean SRA constants for the 40 clinical isolates lead to all strains being interpreted as susceptible. SRA calculations enable laboratories to set up calibrated disk tests with species-related and laboratory-specific interpretations.

Calibration of the disk diffusion test for trovafloxacin susceptibility testing of four anaerobic species.

OBJECTIVES: To study trovafloxacin susceptibility among clinical isolates of four anaerobic bacterial species using minimum inhibitory concentrations (MIC) determinations, E test assays and disk diffusion test results and to calibrate the disk diffusion method for these species using single strain regression analysis (SRA). METHODS: One-hundred and eighty-seven clinical isolates of four anaerobic bacterial species were included. Trovafloxacin MIC determinations were performed using the agar dilution technique and MIC estimations using the E test. The disk diffusion test was performed according to Swedish Reference Group for Antibiotics standardization. NCCLS limits for susceptibility categories were applied. SRA was performed using 1, 3, 10, 30, and 100 microg trovafloxacin disk contents and ATCC control strains. The regression lines obtained permitted the calculation of zone equivalents to MIC limits as well as an evaluation of various disk potencies. RESULTS: Trovafloxacin susceptibility (S + I) was noted in 98.9, 100, 100, and 97% of Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium perfringens, and Peptostreptococcus magnus strains, respectively, as judged by MIC determinations. Agar dilution and E test estimations gave the same results, but E test values were consistently lower than MIC values by the reference method. Regression lines calculated for the four species using SRA showed different equation constants indicating species-related differences. Interpretive zone diameter breakpoints were calculated for the four species and used for the interpretation of susceptibility. CONCLUSIONS: The disk diffusion test was successfully calibrated for trovafloxacin susceptibility testing of four anaerobic species using single strain regression analysis, SRA. There was a good agreement between the results of MIC-tests and disk testing. Interpretive errors of type I are prone to occur among Bacteroides isolates and might require species-related MIC limits. SRA calculations permitted the testing of the effect of different disk potencies on inhibition zones produced at the interpretive MIC limits. Criteria for the selection of a minimal disk content showed that 5 microg trovafloxacin is sufficient, but a 10 microg disk will safeguard against residual laboratory variation without producing too large inhibition zones for very susceptible strains.

MIC determination of fusidic acid and of ciprofloxacin using multidisk diffusion tests.

OBJECTIVE: To investigate the possibility of estimating the MICs of fusidic acid and ciprofloxacin for bacterial isolates using series of antibiotic disk concentrations in diffusion tests, so-called M-tests. METHODS: Thirty Staphylococcus aureus and S. epidermidis strains were tested for fusidic acid susceptibility. Sixty-one clinical isolates of eight bacterial species were tested for ciprofloxacin susceptibility. Disk diffusion was standardized according to the Swedish reference group for antibiotics (SRGA). For fusidic acid, a series of disks (1.5, 5.0, 15, 50 and 150 microg) was used. Ciprofloxacin was applied in four different diffusion sources (1, 3, 10 and 30 microg) on a single strip, the M-strip, and used. True MIC values were determined using the standardized agar dilution method according to the SRGA. Single-strain regression analysis (SRA) was employed to calculate critical concentration equivalents (Qzero). RESULTS: Fusidic acid and ciprofloxacin critical concentrations were determined for the bacterial isolates. The mean conversion factors for Qzero to yield the true MIC were 2.06 (range 0.34-8.9) for fusidic acid and 2.05 (range 0.37-8.1) for ciprofloxacin. There was a correlation between true MIC values (all MICs expressed as 2 log + 9) and the calculated MIC values (Qzero x conversion factor) for both fusidic acid (R = 0.9822) and ciprofloxacin (R = 0.9696). CONCLUSIONS: MIC values of clinical isolates can be estimated using SRA calculations on zone measurements in disk tests with several concentrations of the antibiotic in diffusion sources.

Etiology of pneumonia, sepsis and meningitis in infants younger than three months of age in Ethiopia.

METHODS: Within a multicenter study coordinated by WHO, an investigation of the etiologic agents of pneumonia, sepsis and meningitis was performed among infants younger than 3 months of age seen at the Ethio-Swedish Children's Hospital in Addis Ababa for a period of 2 years. Of the 816 infants enrolled 405 had clinical indications for investigation. RESULTS: There were a total of 41 isolates from blood cultures from 40 infants. The study showed that the traditionally known acute respiratory infection pathogen Streptococcus pneumoniae was most common in this extended neonatal age group, found in 10 of 41 blood isolates. Streptococcus pyogenes was a common pathogen in this setting (9 of 41 blood isolates), whereas Salmonella group B was found in 5 of 41 isolates. Streptococcus agalactiae, which is a common pathogen in developed countries, was absent. A study of the susceptibility pattern of these organisms suggests that a combination of ampicillin with an aminoglycoside is adequate for initial treatment of these serious bacterial infections, but the combination is not optimal for the treatment of Salmonella infections. Among 202 infants on whom immunofluorescent antibody studies for viruses were performed based on nasopharyngeal aspirates, respiratory syncytial virus was found in 57 (28%) infants, and Chlamydia trachomatis was isolated in 32 (15.8%) of 203 infants.