Antimicrobial Minimum Inhibitory Concentration Research Articles

Prediction of Minimum Inhibitory Concentrations of Antimicrobials for Neisseria gonorrhoeae Using Whole-Genome Sequencing.

Bacterial whole-genome sequencing is now increasingly available to researchers, reference laboratories and individual healthcare institutions. It can be possible to predict antimicrobial minimum inhibitory concentrations (MICs) for Neisseria gonorrhoeae using sequencing data, for many antimicrobials within one or two MIC doubling dilutions of the phenotypic value. With emerging rapid sequencing technologies, it may be possible in future to predict antimicrobial resistance faster than existing culture-based methods. Sequencing also provides insights into the genetic mechanisms underlying antimicrobial resistance, their spread in time and space, as well as the molecular epidemiology of the gonococcal strains.

A “Culture” Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood

The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.

Lactobionic acid enhances the synergistic effect of nisin and thymol against Listeria monocytogenes Scott A in tryptic soy broth and milk

Listeria monocytogenes is a Gram-positive opportunistic human pathogen and it remains a significant cause of foodborne illnesses. A variety of natural and synthetic compounds have been studied to inhibit the growth of L. monocytogenes in foods. Antimicrobial combinations with synergistic antilisterial properties can reduce the dose of each antimicrobial, which can be further enhanced by chelating compounds. Therefore, the objective of this study was to determine antilisterial properties of binary or ternary combinations of lactobionic acid (LBA), nisin, and thymol in tryptic soy broth (TSB), 2% reduced-fat milk, and whole milk. The results showed that the minimum inhibitory concentration (MIC) of nisin, thymol and LBA was 125IU/mL, 0.25mg/mL, and 10mg/mL, respectively. The ternary combination was the most effective in reducing MICs of antimicrobials, with the MIC of nisin, thymol, and LBA being 31.25IU/mL, 0.0625mg/mL, and 1.25mg/mL, respectively. In TSB with 0.6% yeast extract, L. monocytogenes grew in individual or binary antimicrobial treatments of 31.25IU/mL nisin, 0.0625mg/mL thymol, and 1.25mg/mL LBA within 24h at 32°C, while it was completely inhibited by the ternary combination. In 2% reduced-fat milk at 21°C, the ternary combination of nisin, thymol, and LBA at respective concentrations of 250IU/mL, 2mg/mL, and 10mg/mL completely inhibited the bacterium to below the detection limit in 72h while >2log (CFU/mL) bacteria was still detected in all the binary combinations after 120h. In whole milk, the combination of 500IU/mL nisin, 2mg/mL thymol, and 10mg/mL LBA reduced bacteria to around 2log (CFU/mL) in 4h at 21°C, and no bacterial recovery was observed after 5 d. This study suggested the potential of the ternary combination of nisin, thymol and LBA for food preservation.

Molecular mechanisms of membrane impermeability in clinical isolates of Enterobacteriaceae exposed to imipenem selective pressure

Intrinsic mechanisms leading to carbapenem-induced membrane impermeability and multidrug resistance are poorly understood in clinical isolates of Enterobacteriaceae. In this study, molecular behaviours during the establishment of membrane impermeability in members of the Enterobacteriaceae family under imipenem selective pressure were investigated. Clinical isolates (n = 22) exhibiting susceptibility to multiple antibiotics or characterised as extended-spectrum β-lactamase (ESBL)- or AmpC-producers were submitted to progressive passages on Mueller–Hinton agar plates containing subclinical concentrations of imipenem [0.5 × the minimum inhibitory concentration (MIC)]. Changes in outer membrane permeability were evaluated by determination of antimicrobial MICs, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and gene expression analysis related to membrane permeability (i.e. omp35-like, omp36-like and acrA) and regulatory mechanisms (i.e. marA and ompR) by quantitative reverse transcription PCR. Following imipenem induction, 73% of isolates showed increased carbapenem MICs by ≥2 doubling dilutions. At an early stage of treatment, imipenem modulated the expression of porins and efflux pump genes, represented by a reduction of 78% in omp36-like and a two-fold increase in acrA expression. Transcriptional factors marA and ompR were also affected by imipenem induction, increasing mRNA expression by 14- and 4-fold, respectively. High marA expression levels were associated with higher values of acrA expression. These results suggest that imipenem is an important factor in the development of an adaptive response to carbapenems by regulating key genes involved in the control of efflux pumps and porins, which could lead to a multidrug-resistant profile in clinical isolates, contributing to possible treatment failure.

Isolation Method (Direct Plating or Enrichment) does Not Affect Antimicrobial Susceptibility of Campylobacter from Chicken Carcasses

AbstractTo determine if Campylobacter isolation method influenced antimicrobial susceptibility results, the minimum inhibitory concentrations (MIC) of nine antimicrobials were compared for 291 pairs of Campylobacter isolates recovered from chicken carcass rinse samples using direct plating and an enrichment method. Among the isolates 64.1% were C. jejuni, 35.7% were C. coli, and 0.2% were C. lari. Direct plating yielded significantly less (P < 0.05) C. coli (21.3%) compared to sample enrichment (50.2%). Antimicrobial resistance was most common for tetracycline (41.4%), nalidixic acid (26.3%), and ciprofloxacin (25.9%). Significantly more (P < 0.05) C. coli were resistant to nalidixic acid, ciprofloxacin, gentamicin, azithromycin, and erythromycin as compared to C. jejuni isolates. Nonparametric bootstrap analysis of antimicrobial MICs showed no significant differences between direct plating and the enrichment method for any of the antimicrobials tested. These data indicate that bacterial isolation method can bias Campylobacter species recovery. However, isolation method did not significantly affect antimicrobial susceptibility results of C. jejuni or C. coli recovered from broiler carcasses.Practical ApplicationsTo monitor trends in food safety and public health, antimicrobial susceptibility testing of Campylobacter derived from poultry products and infected patients has become common practice in both regulatory food safety and public health programs. Various methods have been employed for Campylobacter recovery including direct plating for enumeration of contamination levels and enrichment protocols for detection of low numbers or injured cells. This study was conducted to determine if the method of Campylobacter isolation from chicken carcass rinsate, direct plating or enrichment, influences antimicrobial susceptibility testing results.

Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China.

Mycoplasma bovis has spread widely throughout the world via animal movement and has become an important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations of antimicrobials for Mycoplasma bovis have not been studied in China. The objective of this study was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolated from young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45 bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible or had medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant to macrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in the rrnA operon in domain V of 23S rRNA was observed in strains that were resistant to macrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in the resistance of Mycoplasma bovis strains to macrolides.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Prise en charge des accidents vasculaires cérébraux dans un service de médecine interne de la banlieue de Dakar

Co-trimoxazole is widely used in treatment of paediatric pneumonia in developing countries, but drug resistance may decrease its effectiveness. We studied the effectiveness of co-trimoxazole compared with that of amoxycillin in pneumonia therapy, and assessed the clinical impact of co-trimoxazole resistance.We recruited 595 children, aged 2–59 months, with non-severe or severe pneumonia (WHO criteria) diagnosed in the outpatient wards of two urban Pakistan hospitals. Patients were randomly assigned on a 2:1 basis co-trimoxazole (n=398) or amoxycillin (n=197) in standard WHO doses and dosing schedules, and were monitored in study wards. The primary outcome was inpatient therapy failure (clinical criteria) or clinical evidence of pneumonia at outpatient follow-up examination.There were 92 (23%) therapy failures in the co-trimoxazole group and 30 (15%) in the amoxycillin group (p=0·03)—26 (13%) versus 12 (12%) among children with non-severe pneumonia (p=0·856) and 66 (33%) versus 18 (18%) among those with severe pneumonia (p=0·009). For patients with severe pneumonia, age under 1 year (p=0·056) and positive chest radiographs (p=0·005) also predicted therapy failure. There was no significant association between antimicrobial minimum inhibitory concentration and outcome among bacteraemic children treated with co-trimoxazole.Co-trimoxazole provided effective therapy in non-severe pneumonia. For severe, life-threatening pneumonia, however, co-trimoxazole is less likely than amoxycillin to be effective.

LukSF-PVinStaphylococcus aureusKeratitis Isolates and Association With Clinical Outcome

To determine the prevalence, genetic diversity, and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton-Valentine leukocidin, in Staphylococcus aureus isolates from cases of bacterial keratitis in the United Kingdom. Multiplex PCRs investigating carriage of lukSF-PV and mecA were performed on S. aureus isolates from patients. The lukSF-PV operon was sequenced to investigate its diversity, and multilocus sequence typing to test for a clonal relationship between lukSF-PV isolates. Antimicrobial minimum inhibitory concentrations (MICs) and clinical outcome data were compared for isolates characterized as lukSF-PV+ve, mecA+ve, and lukSF-PV/mecA-ve. Of 95 isolates, 9 (9.5%) were lukSF-PV+ve, 9 (9.5%) mecA+ve, and 1 was positive for both. Five single nucleotide polymorphisms were found in lukSF-PV genes of seven strains. There was no significant difference between the MICs of lukSF-PV/mecA-ve and lukSF-PV+ve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecA+ve isolates had significantly higher mean MICs to meropenem and fluoroquinolones (P < 0.05). There were nonsignificant trends for healing and treatment times, ulcer and scar size, and overall clinical score to be greater in the lukSF-PV+ve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PV+ve isolates with an odds ratio of 7.8 (95% CI 1-42, P = 0.018) for patients requiring surgery. lukSF-PV+ve isolates were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus-associated keratitis.

N-Vitro Antimicrobial Activity of Membrane-Acting Antibiotics Action Against Streptococci

Streptococcus equi subS. equi a group C streptococcus is a gram-positive bacterium and is the causative agent of one of the most prevalent diseases of horses known as strangles, although, a live attenuated S. equi vaccine for prevention of the strangles has been introduced, there are presently still no proven vaccine against S. equi. In the effort to develop an effective vaccine against S. equi, the minimum inhibitory concentrations of antimicrobials acting bacterial membrane - triclosan and 4-Methylumbelliferone (4-MU) on S. equi have been investigated. These were determined by serial dilution technique on three strains of the equine S. equi: 4047, K3, and NCTC 9682. In addition, sensitive disk test on solid media were also carried out. The result of this study demonstrated that the three strains of S. equi are very sensitive to triclosan as they inhibit certain membrane functions. However, these strains are very resistant to 4-MU. Further research to investigate the mechanism of interaction between these antimicrobial agents and the bacterial membrane components is required.

Characterization of coagulase-negative staphylococcus species from cows’ milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n=366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMérieux, Marcy l’Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows’ environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.

ANTIMICROBIAL RESISTANCE IN ENTEROBACTERIACEAE FROM INTENSIVELY-REARED APPARENTLY HEALTHY AND DISEASED POULTRY IN ABEOKUTA, NIGERIA

The emergence and wide-spread dissemination of antimicrobial resistant bacteria strains is a global phenomenon of great public health and economic implications. Antimicrobial resistance was investigated in enterobacteriaceae isolated from apparently healthy and diseased poultry birds using the broth micro-dilution method to determine antimicrobial minimum inhibitory concentration (MIC). In all, 504 bacterial isolates including Escherichia coli (471), Klebsiella spp (28) and Salmonella enterica isolates (5) were studied. The isolates were resistant to ampicillin (88.5%), chloramphenicol (62.3%), ciprofloxacin (74.8%), enrofloxacin (81.0%), neomycin (83.9%), norfloxacin (78.8%), streptomycin (91.3%) and tetracycline (83.3%). The geometric mean MIC (µg/µL) of tested antimicrobials for enterobacteriaceae is as follows: ampicillin (102.5), chloramphenicol (48.4), ciprofloxacin (19.1), enrofloxacin (34.5), neomycin (47.7), norfloxacin (24.5), streptomycin (142.2) and tetracycline (62.5). Although rates of resistance to ampillin, streptomycin and tetracycline were similar among isolates from apparently healthy and diseases birds, resistance to chloramphenicol, ciprofloxacin, enrofloxacin, neomycin and norfloxacin were significantly higher (p<0.05) in isolates from diseased chickens than in those from apparently healthy chickens. The high rates of antimicrobial resistance in bacteria may contribute to the persistence of pathogens in poultry flock and ineffectiveness of antimicrobial chemotherapy during disease outbreaks.Â

Antimicrobial Susceptibility of Erysipelothrix rhusiopathiae Isolated from Pigs in Southern Japan with a Modified Agar Dilution Method

The determination of antimicrobial minimum inhibitory concentration (MIC) in Erysipelothrix rhusiopathiae by using the agar dilution method has not been covered by the Clinical and Laboratory Institute (CLSI). Only the broth microdilution method has been outlined. This report describes a modification of the agar dilution procedure for E. rhusiopathiae using Trypto-soy agar supplemented with 0.1% Tween 80 and incubation in ambient air at 37 degrees C for 24 hr. The MICs of the assay were in agreement with those of the broth microdilution method recommended by the CLSI. Antimicrobial susceptibility test was performed using this method for 149 E. rhusiopathiae isolates from 2 meat processing plants in Kagoshima Prefecture during the period of April 2004 to March 2005. The number of strains resistant to oxytetracycline, erythromycin, lincomycin, ofloxacin and enrofloxacin were 56 (37.6%), 4 (2.7%), 18 (12.1%), 21 (14.1%) and 19 (12.8%), respectively. All strains were susceptible to ampicillin.

Antimicrobial drug resistance of Escherichia coli isolated from poultry abattoir workers at risk and broilers on antimicrobials

Antimicrobial usage in food animals increases the prevalence of antimicrobial drug resistance among their enteric bacteria. It has been suggested that this resistance can in turn be transferred to people working with such animals, e.g., abattoir workers. Antimicrobial drug resistance was investigated for Escherichia coli from broilers raised on feed supplemented with antimicrobials, and the people who carry out evisceration, washing and packing of intestines in a high-throughput poultry abattoir in Gauteng, South Africa. Broiler carcasses were sampled from 6 farms, on each of which broilers are produced in a separate 'grow-out cycle'. Per farm, 100 caeca were randomly collected 5 minutes after slaughter and the contents of each were selectively cultured for E. coli. The minimum inhibitory concentration (MIC) of each isolate was determined for the following antimicrobials: doxycycline, trimethoprim, sulphamethoxazole, ampicillin, enrofloxacin, fosfomycin, ceftriaxone and nalidixic acid. The same was determined for the faeces of 29 abattoir workers and 28 persons used as controls. The majority of isolates from broilers were resistant, especially to antimicrobials that were used on the farms in the study. Overall median MICs and the number of resistant isolates from abattoir workers (packers plus eviscerators) tended to be higher than for the control group. However, no statistically significant differences were observed when the median MICs of antimicrobials used regularly in poultry and percentage resistance were compared, nor could an association between resistance among the enteric E. coli from packers and those from broilers be demonstrated.

Effect of an Efflux Pump Inhibitor on the Function of the Multidrug Efflux Pump CmeABC and Antimicrobial Resistance inCampylobacter

CmeABC, a multidrug efflux pump, contributes to the resistance of Campylobacter to a broad range of antimicrobials. We hypothesize that an efflux pump inhibitor (EPI) may inhibit the function of CmeABC and control antibiotic resistance in Campylobacter. In this study, we examined the effect of EPI Phe-Arg beta-naphthyl-amide dihydrochloride (MC-207,110) on the susceptibility of Campylobacter to various antimicrobials. The presence of the EPI resulted in a 2- to 2048-fold reduction in the minimum inhibitory concentration (MIC) of antimicrobials known to be substrates of the CmeABC pump in all Campylobacter strains. Both intrinsic and acquired resistance of C. jejuni to erythromycin was decreased drastically (64- to 128-fold reduction in the MIC) in the presence of the EPI while the MICs of fluoroquinolones were only slightly decreased (2- to 4-fold). Examination of 57 Campylobacter isolates from various origins further demonstrated that MC-207,110 decreased the MICs of erythromycin (2- to 512-fold) in all isolates. Compared to wild-type strains, the isogenic CmeB mutants displayed smaller magnitudes of reduction in the MICs of antimicrobials in the presence of the EPI, indicating the inhibitory effect of the EPI is primarily CmeABC-dependent. The inhibitory effect of MC-207,110 was also dose-dependent, and as little as 0.5 microg/mL of the EPI resulted in a decreased MIC for erythromycin in C. jejuni. More importantly, the presence of MC-207,110 decreased the frequency of emergence of erythromycin-resistant mutants in C. jejuni (<10(11), well below the normal frequency of approximately 10(8)). Together, these findings indicate that EPI MC-207,110 inhibits the function of CmeABC efflux pump and potentiates the activity of antibiotics against Campylobacter. Inhibition of CmeABC by EPI is a promising approach in combating antibiotic resistance of Campylobacter in humans and animal reservoirs.

Comparison of primary and secondary antimicrobial minimum inhibitory concentrations for Helicobacter pylori isolated from Korean patients

In this study we assessed minimum inhibitory concentration (MIC) values and resistance rates of several antibiotics in 65 primary and 324 secondary Helicobacter pylori isolates from Korean patients. Primary resistance to amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, ciprofloxacin, levofloxacin and moxifloxacin was 18.5%, 13.8%, 66.2%, 12.3%, 32.3%, 33.8%, 21.5% and 21.5%, respectively. Secondary resistance was 31.3%, 85.1%, 70.1%, 0%, 89.6%, 35.8%, 32.8% and 32.8%, respectively. Sequence analysis of pbp1A in H. pylori strains with an amoxicillin MIC ≥2 μg/mL revealed C206T (Asp69 → Val), C1667G (Thr556 → Ser), A1684T (Asn562 → Tyr), A1777G (Thr593 → Ala) and C1798A (Pro600 → Thr) substitutions. Eleven (16.4%) of 67 treatment failures showed mixed infections with antibiotic-susceptible and -resistant H. pylori. The most common multidrug resistance profile was to clarithromycin, metronidazole and azithromycin. These results indicate that MIC values of secondary isolates were higher than those of primary isolates and that resistance to amoxicillin is probably mediated through mutations in pbp1A.

Analysis of plasmid profiles of Aeromonas salmonicida isolates by pulsed field gel electrophoresis

Plasmid constitutions of Aeromonas salmonicida isolates were characterised by flat-bed and pulsed field gel electrophoresis. Resolution of plasmids by pulsed field gel electrophoresis was greater and more consistent than that achieved by flat-bed gel electrophoresis. The number of plasmids separated by pulsed field gel electrophoresis varied between A. salmonicida isolates, with five being the most common number present in the isolates used in this study. Plasmid profiles were diverse and the reproducibility of the distances migrated facilitated the use of principal components analysis for the characterisation of the isolates. Isolates were grouped according to the number of plasmids supported. Further principal components analysis of groups of isolates supporting five and seven plasmids showed a spatial separation of plasmids based upon distance migrated. Principal components analysis of plasmid profiles and antimicrobial minimum inhibitory concentrations could not be correlated suggesting that resistance to antimicrobial agents is not associated with either one plasmid or a particular plasmid constitution.

Statistical properties and inference of the antimicrobial MIC test

A common method for measuring the drug-specific minimum inhibitory concentration (MIC) of an antibacterial agent is via a two-fold broth dilution test known as the MIC test. Because this procedure implicitly rounds data upward, inference based on unadjusted measurements is biased and overestimates bacterial resistance to a drug. We detail this test procedure and its associated bias, which, in many cases, has an expected value of approximately 0.5 on the log(2) scale. In addition, new bias-corrected estimates of resistance are proposed. A numeric example is used to illustrate the extent to which the traditional resistance estimate can overestimate the true proportion of resistant strains, a phenomenon which is remedied by using the proposed estimates.

Presence of a mutation in ponA1 of Neisseria gonorrhoeae in numerous clinical samples resistant to various β-lactams and other, structurally unrelated, antimicrobials

The number of resistant strains in patients with Neisseria gonorrhoeae urethritis has been increasing, making effective treatment difficult. Chromosomally mediated penicillin-resistant N. gonorrhoeae arise through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. To understand the occurrence of penicillin resistance in patients with N. gonorrhoeae infection, we performed this study. In addition, we studied minimum inhibitory concentrations (MICs) of antimicrobials against N. gonorrhoeae strains. We measured the MICs of penicillin G, other beta-lactams, and other kinds of antimicrobials against 53 clinical N. gonorrhoeae isolates from male patients with urethritis in Hyogo and Osaka, Japan. The ponA genes, encoding PBP 1 of these isolates, were sequenced. Of the 53 isolates tested, 41 strains showed some resistance to penicillin G. A mutation in the ponA (ponA1) gene was identified in 46 isolates. There was a tendency that ponA mutant (ponA1) in N. gonorrhoeae led to higher antimicrobial MICs of beta-lactam antimicrobial agents (including penicillin) than those of non-ponA mutants. However, we found lower than expected MICs of penicillin and beta-lactams even in ponA mutants. Therefore, we consider that detailed investigations for the further understanding of the effect of other genes, such as penC (which is reported to be related to ponA1 in achieving high-level penicillin resistance) should be our next step.

Burkholderia cepacia complex genomovars: utilization of carbon sources, susceptibility to antimicrobial agents and growth on selective media.

To investigate the relationship between genomovar status and carbon source utilization, antibiotic susceptibility and growth ability on selective media of 142 clinical and environmental Burkholderia cepacia complex (Bcc) isolates belonging to all nine genomovars. Carbon source utilization and growth on selective media were tested by agar plate multipoint inoculation. Antimicrobial minimum inhibitory concentration (MIC) values were determined by agar dilution. Of all carbon sources, l-arabinose was most frequently utilized, supporting growth of 90% of all isolates. Burkholderia cepacia genomovar VI failed to utilize azelaic acid, penicillin G, phtalate, salicin and tryptamine. Overall, B. vietnamiensis and B. anthina were most susceptible and B. cepacia genomovar VI most resistant to antimicrobial agents. Burkholderia cepacia selective agar (BCSA) and the Mast B. cepacia medium supported growth of Bcc isolates most efficiently. This study demonstrates phenotypic heterogeneity within the Bcc. Some trends can be observed at the genomovar level, but only B. cepacia genomovar VI could be differentiated unambiguously on the basis of its inability to grow on PCAT. This work provides an update on some differential phenotypic characteristics of all nine Bcc genomovars.