Genotypic variation in the Bordetella pertussis virulence factors pertactin and pertussis toxin in historical and recent clinical isolates in the United Kingdom

The reemergence of pertussis has been reported in several countries despite high vaccination coverage. Studies in The Netherlands and Finland have investigated polymorphism in the genes coding for two important virulence factors of Bordetella pertussis, pertactin and pertussis toxin, and identified the emergence and subsequent dominance in circulating strains of pertactin and toxin variants not found in the whole-cell vaccine (WCV). The study described here investigated whether such variation had occurred in the United Kingdom, which presently has low levels of pertussis. Sequence analysis of the genes for pertactin (prnA) and the pertussis toxin S1 subunit (ptxA) among isolates of B. pertussis from 285 United Kingdom patients, from 1920 to 1999, revealed three prnA variants, prnA(1), prnA(2), and prnA(3), and two ptxA variants, ptxA(1) and ptxA(2), showing differences in nucleic acid sequence. The proportion of pertactin gene types not included in the United Kingdom WCV, i.e., prnA(2) and prnA(3), has increased in recent years and was found in 21 of 86 (24%) strains from the 1980s and 56 of 105 (53%) strains from the 1990s. To date, the presence of these nonvaccine prnA types has not been associated with a resurgence of pertussis in the United Kingdom. The distribution of prnA and ptxA types in The Netherlands, Finland, and the United Kingdom in the 1990s is distinct. The most striking difference in the United Kingdom isolates is that all 105 of the most recent circulating strains (from 1998 to 1999) are of a pertussis toxin type found in the United Kingdom WCV, i.e., ptxA(1).     

Antibiotic susceptibility testing of Bordetella pertussis

A tube dilution test to evaluate the effectiveness of antibiotics against Bordetella pertussis is described. Five B. pertussis strains, including a well-characterized research strain and four fresh clinical isolates, were tested with several antibiotics. Erythromycin showed the highest in-vitro activity of the antibiotics tested. A concentration of 0.12 microgram/ml was bacteriostatic for all strains, while 2 microgram/ml was bactericidal. Minimal inhibitory and minimal bactericidal concentrations for ampicillin by tube tests were found to be higher than values previously reported for agar plate tests.     

Application of the Etest to the antimicrobial susceptibility testing of Mycobacterium marinum clinical isolates.

Mycobacterium marinum, a well-recognized cutaneous pathogen, is usually treated by chemotherapy without available standardized in vitro susceptibility testing information. In this study, we have attempted to apply the stable-gradient method (Etest; AB Biodisk, Solna, Sweden) to susceptibility testing of M. marinum in order to assess the activities of eight antimicrobial agents against 60 recent clinical strains of M. marinum collected from 10 geographic sites within the United States. Two plated media (5% sheep blood Mueller-Hinton agar and Middlebrook 7H11 agar) were compared, and 7H11 agar was found to be superior in supporting the growth of all strains. Four reference strains of M. marinum were tested on five occasions with eight drugs (160 tests) in order to evaluate Etest reproducibility. Results were observed to be within 1 log2 dilution of the all-test median MIC for 97.5% of the Etests. Our MIC results for the 60 strains clearly demonstrate the best in vitro potency against M. marinum isolates to be as follows (rank order): trimethoprim-sulfamethoxazole (MIC at which 90% of the isolates are inhibited [MIC90], 0.25 and 4.25 microg/ml, respectively) = ethambutol > clarithromycin (MIC90, 1 microg/ml) > minocycline = doycycline (MIC90, 4 microg/ml) > amikacin (MIC90, 8 microg/ml). Rifampin was only marginally active against the M. marinum strains tested (MIC90, at the National Committee for Clinical Laboratory Standards) breakpoint of 1 microg/ml), and ciprofloxacin was not active (MIC90, 8 microg/ml). These data should enhance the empiric drug selection for contemporary M. marinum infections and also provide evidence that the Etest can be utilized to guide chemotherapy with alternative agents.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Comparison of Etest and agar dilution method for antimicrobial susceptibility testing of Flavobacterium isolates.

The Etest was evaluated as a possible alternative to the standard agar dilution method for susceptibility testing of nine antimicrobial agents against Flavobacterium species. In studies of 100 clinical isolates, the agreement between the MICs (+/-1 log2 dilution) obtained by the two methods was acceptable for cefotaxime, ceftazidime, amikacin, minocycline, ofloxacin, and ciprofloxacin (> 90%). Conversely, the agreement between the results obtained for piperacillin was limited (84%). The overall agreement was 92.5%.     

Antimicrobial susceptibility testing of veterinary clinical isolates with the Sceptor System.

The Sceptor System (Becton Dickinson) was compared with an agar dilution method for antimicrobial susceptibility testing of veterinary clinical isolates. The results indicate that the Sceptor System may be used to test gram-positive and fastidious gram-negative bacteria.     

Evaluation of Etest for susceptibility testing of multidrug-resistant isolates of Mycobacterium tuberculosis

To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques for Mycobacterium tuberculosis are needed, especially in regions with multidrug resistance. Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for M. tuberculosis in 5 to 10 days from a culture grown at low cost. The performance of Etest was compared to that of the reference proportion method, using 95 M. tuberculosis clinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method. Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin. This study supports the utility of Etest for timely detection of drug resistance in M. tuberculosis and for use in tuberculosis control programs.     

Antigenic divergence of Bordetella pertussis isolates in Taiwan

In recent studies, antigenic divergence has been observed in Bordetella pertussis circulating isolates. We collected 80 Bordetella pertussis isolates in Taiwan from 1998 to 2004 and analyzed them using a combination of pulsed-field gel electrophoresis (PFGE) and sequencing of the ptxS1 and prn genes. The incidence of pertussis increases every 3 years, and most of the isolates prevalent since 1998 have expressed nonvaccine ptxS1A and prn2 alleles. Through PFGE analysis, all isolates could be classified into four major groups, and the incidence of these groups exhibited a correlation with the prn allele expressed by the isolates. We found that PFGE is more discriminative than gene sequencing, since it could divide the isolates expressing the prn2 allele into two groups: one group circulating from 1998 to 2001 and another group circulating from 2001 to 2004. The transition between the two groups in 2000 coincided with an outbreak of 326 cases. This research indicates that the antigenic divergence of B. pertussis circulating isolates has evolved over time in Taiwan. Such information will have implications for vaccine policy in Taiwan.     

Comparison of PCR detection of mecA with agar dilution and Etest for oxacillin susceptibility testing in clinical isolates of coagulase-negative staphylococci

Oxacillin-resistant staphylococci are heterogeneous in their expression of resistance to beta-lactam antibiotics. Different recommendations regarding screening methods for routine use have been published. In this study, the susceptibility to oxacillin of 232 coagulase-negative staphylococci (CoNS) was determined by agar dilution, Etest and presence of the mecA gene. When an oxacillin resistance breakpoint of > or = 0.5 mg/L was used, the sensitivity and specificity for agar dilution were 97.6% and 100%, and those for Etest were 100% and 95.4%. The current National Committee for Clinical Laboratory Standards oxacillin breakpoint recommendation will categorise accurately the CoNS species encountered commonly.     

Survey of amphotericin B susceptibility of Candida clinical isolates determined by Etest.

BACKGROUND AND PURPOSE: The minimal inhibitory concentrations (MICs) of amphotericin B (AmB) determined by the National Committee for Clinical Laboratory Standards (NCCLS; NCCLS document M27-A) broth dilution method are in a relatively narrow ranges and this may lead to underestimation of the AmB-resistant rate in clinical isolates. We evaluated in vitro susceptibility of clinical isolates of Candida spp. to AmB using Etest and determined the distribution of AmB MICs in different species. METHODS: We used the Etest (AB Biodisk, Solna, Sweden) to evaluate the MICs of Candida isolates randomly collected during 2001-2003 in a teaching hospital. RESULTS: Of the 572 isolates evaluated, Candida albicans (50.7%) was the most common species, followed by Candida tropicalis (23.9%), Candida parapsilosis (13.1%), Candida glabrata (9.4%), Candida krusei (1.9%), and Candida guilliermondii (0.9%). The majority of isolates were from blood (85%). The minimal concentrations of AmB required to inhibit 50%/90% of the isolates (MIC(50)/MIC(90)) were 0.19/0.38 microg/mL for C. krusei, 0.125/0.38 microg/mL for C. glabrata, 0.094/0.25 microg/mL for C. tropicalis, 0.032/0.19 microg/mL for C. albicans, 0.016/0.125 microg/mL for C. parapsilosis, and 0.023/0.032 microg/mL for C. guilliermondii. Only 1 blood isolate of C. glabrata was resistant to AmB (MIC > or =1 microg/mL) [0.17%]. 18.2% of isolates were less susceptible to AmB (MIC > or =0.19 microg/mL) with the highest rates for C. krusei (63.6%), followed by C. glabrata (37.0%), C. tropicalis (29.9%), C. albicans (11.0%), C. parapsilosis (5.3%), and C. guilliermondii (0%). More isolates collected from patients with hematologic malignancy were less susceptible to AmB than those collected from those with other diseases (30.5% vs 15.4%, p<0.05). CONCLUSION: This study demonstrated that AmB resistance remains rare at this hospital in Candida clinical isolates despite increasing use of this agent during the past 4 decades.     

Antimicrobial susceptibility pattern of clinical isolates of non-fermentative bacteria

152 nonfermentative bacteria were isolated from a total number of 965 clinical samples processed routinely in the laboratory of Microbiology Department, M.K.C.G Medical College in South Orissa accounting to a prevalence rate of 15.75%. Pseudomonas spp. (both pigmented and non-pigmented strains) were isolated in maximum percentage (73.6%) followed by Acinetobacter spp. (19.7%) and Alkaligenes faecalis (4.6%). Rarely encountered species were Eikenella corrodens (1.3%) and Stenotrophomonas maltophila (0.6%). Pus from various sites was the major source (116; 76%). 81% of all isolates were sensitive to amikacin and 74% to ofloxacin. Sensitivity to cefotaxime, ciprofloxacin, tobramycin, gentamicin and netlimycin ranged from 53% to 68%. Least effective drugs were carbenicillin and ceftriaxone (48% each).     

Antimicrobial susceptibility of clinical isolates from earthquake victims in Wenchuan

On 12 May 2008, an earthquake measuring 8.0 on the Richter scale struck Wenchuan County, Sichuan, China. Between 12 May and 11 June, 1823 victims were hospitalized in West China Hospital. These patients were severely injured, and most of their wounds were contaminated. Here, the results of bacteriological identification and antibiotic susceptibility testing of 725 non-duplicate isolates from earthquake victims are presented. Gram-negative bacilli were most frequently isolated (71.3%). Only 18.9% of isolates were Gram-positive bacteria; Candida spp. accounted for 9.7%, and Gram-negative cocci for 0.1%. After anaerobic culture, four Clostridium sordellii strains and one Clostridium bifermentans strain were isolated from deep wounds. Specimen culture from earthquake victims revealed a spectrum of pathogens and antibiotic susceptibilities that was different from that usually encountered in West China Hospital, especially concerning methicillin-resistant Staphylococcus aureus , extended-spectrum β-lactamase producers, and multidrug-resistant (MDR) non-fermenting Gram-negative bacilli. The pathophysiology of the injuries in earthquake victims was different from that in the patients who were not earthquake victims. A combination of environmental bacteria with a high proportion of Gram-negative bacteria was often observed in the earthquake victims. Approximately 26% of all earthquake victims were shown to be carriers of MDR microorganisms. Therefore, appropriate microbiological assessment upon admission, and identification of patients to be put in quarantine, is of paramount importance.     

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.     

Evaluation of disk diffusion and Etest compared to broth microdilution for antifungal susceptibility testing of posaconazole against clinical isolates of filamentous fungi

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of posaconazole against 146 clinical isolates of filamentous fungi. By using provisional breakpoints for comparison purposes only, categorical agreement between the results of the agar-based methods and those of broth microdilution were 96 to 98%, with no very major errors. These agar-based methods hold promise as simple and reliable methods for determining the posaconazole susceptibilities of filamentous fungi.     

Antimicrobial susceptibility patterns of clinical isolates of Staphylococcus aureus in Nepal

Isolates of Staphylococcus aureus (n = 117) from patients attending a tertiary care centre in western Nepal were tested for susceptibility to penicillin, oxacillin, gentamicin, erythromycin and ciprofloxacin. Eighteen (15.4%) were methicillin-resistant. Susceptibility among methicillin-resistant isolates varied from 0% (penicillin) to 16.6% (erythromycin and gentamicin), but varied among methicillin-susceptible isolates from 39.4% (penicillin) to 97.0% (ciprofloxacin). Fourteen (77.8%) of the methicillin-resistant isolates were resistant to all agents tested. Implementation of an appropriate antibiotic policy would reduce the risk of further development of antimicrobial resistance.     

Prevalence and Molecular Characterization of Pertactin-Deficient Bordetella pertussis in the United States

Pertussis has shown a striking resurgence in the United States, with a return to record numbers of reported cases as last observed in the 1950s. Bordetella pertussis isolates lacking pertactin, a key antigen component of the acellular pertussis vaccine, have been observed, suggesting that B. pertussis is losing pertactin in response to vaccine immunity. Screening of 1,300 isolates from outbreak and surveillance studies (historical isolates collected from 1935 up to 2009, isolates from the 2010 California pertussis outbreak, U.S. isolates from routine surveillance between 2010-2012, and isolates from the 2012 Washington pertussis outbreak) by conventional PCR and later by Western blotting and prn sequencing analyses ultimately identified 306 pertactin-deficient isolates. Of these pertactin-deficient strains, 276 were identified as having an IS481 in the prn gene (prnIS481 positive). The first prnIS481-positive isolate was found in 1994, and the next prnIS481-positive isolates were not detected until 2010. The prevalence of pertactin-deficient isolates increased substantially to more than 50% of collected isolates in 2012. Sequence analysis of pertactin-deficient isolates revealed various types of mutations in the prn gene, including two deletions, single nucleotide substitutions resulting in a stop codon, an inversion in the promoter, and a single nucleotide insertion resulting in a frameshift mutation. All but one mutation type were found in prn2 alleles. CDC 013 was a predominant pulsed-field gel electrophoresis (PFGE) profile in the pertactin-positive isolates (203/994) but was found in only 5% (16/306) of the pertactin-deficient isolates. Interestingly, PFGE profiles CDC 002 and CDC 237 represented 55% (167/306) of the identified pertactin-deficient isolates. These results indicate that there has been a recent dramatic increase in pertactin-deficient B. pertussis isolates throughout the United States.     

Antigenic divergence between Bordetella pertussis clinical isolates from Moscow, Russia, and vaccine strains

We analyzed temporal changes in the frequencies of the ptxA, prn, fim2, and fim3 alleles in Bordetella pertussis strains isolated from pertussis patients in Moscow, Russia, from 1948 to 2004. The three strains used for the whole-cell vaccine harbored the alleles ptxA2, ptxA4, prn1, fim2-1, and fim3A. Vaccine-type alleles of ptxA (ptxA2 and ptxA4) were characteristic for all prevaccination strains and for 96% of the strains isolated in the 1960s and 1970s. At the beginning of the 1970s, ptxA2 and ptxA4 were replaced by the ptxA1 allele. In the 1980s and to the present, strains with ptxA1 were predominant in the B. pertussis population. All prevaccination strains harbored the prn1 allele, which corresponds to the vaccine-type allele. In subsequent years, the proportion of strains with the prn1 allele decreased and the proportion of prn3 and prn2 strains increased. From 2002 to 2004 strains with prn2 or prn3 were predominant in the B. pertussis population. The vaccine-type alleles fim2-1 and fim3A were found in all prevaccination strains and in 92% of the strains isolated from 1960 to 1989. The fim2-2 and fim3B alleles were first observed at the beginning of the 1980s. In subsequent years, these strains became predominant. Together with waning immunity, the antigenic divergence between vaccine strains and clinical isolates observed in the Moscow area may explain the persistence of pertussis, despite the high rates of vaccine coverage. The results demonstrate that the selection of B. pertussis strains for vaccine manufacturing must be based on a thorough study of the B. pertussis population.     

Evaluation of Etest and disk diffusion methods compared with broth microdilution antifungal susceptibility testing of clinical isolates of Candida spp. against posaconazole

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of 2,171 clinical isolates of Candida spp. against posaconazole. By using provisional breakpoints for comparison purposes only, the categorical agreement between the agar-based methods and broth microdilution results ranged from 93 to 98%, with <1% very major errors. The essential agreement (within 2 well dilutions) between the Etest and broth microdilution methods was 94%. These agar-based methods hold promise as simple and reliable methods for determination of the posaconzole susceptibilities of Candida spp.