Fluoroquinolone resistance in Haemophilus influenzae from nursing home residents in Taiwan: correlation of MICs and mutations in QRDRs.

AIMS: To study the association between number and positions of mutations with MICs of fluoroquinolone non-susceptible Haemophilus influenzae. METHODS AND RESULTS: More than 40% of 48 H. influenzae isolated from nursing home residents were not susceptible to fluoroquinolone. Amino acid changes in the quinolone resistance determining regions, and correlation with MICs and inhibition zone diameters were analysed. All isolates with reduced susceptibility to fluoroquinolones (MIC ≥0·125 µg ml-1 ) had at least one mutation in gyrA at position 84 and were resistant to nalidixic acid. Compared to isolates with reduced susceptibility, resistant isolates were associated with mutations in gyrA at positions 88 and 134, and in parC at position 88 (P < 0·001). Inhibition zone diameter for nalidixic acid disk ≥23 mm may detect susceptible isolates. CONCLUSIONS: Reduced susceptibility to fluoroquinolones was associated with mutations at position 84 in gyrA. A further increase in fluoroquinolone MIC was associated with mutations in gyrA at positions 88 and 134, and parC at position 88. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to limited resistant H. influenzae strains, prior studies on association between positions of mutations and fluoroquinolone MICs were inconclusive. The comparison of mutations between isolates with susceptibility, reduced susceptibility and high resistance supported the importance of the present study.

Carriage rates of methicillin-resistant Staphylococcus aureus (MRSA) depend on anatomic location, the number of sites cultured, culture methods, and the distribution of clonotypes.

The present study was carried out to determine how active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) could be improved by the use of enrichment broth and the inclusion of extra-nasal sites with nares cultures. Molecular typing was also performed to identify colonization by single or multiple strains. Surveillance cultures for MRSA were obtained from 650 patients on admission to a medical and surgical intensive care unit (ICU) in Taiwan. MRSA was detected on directly plated vs. broth-enrichment cultures in any site at 10.0% vs. 24.2%, nares 8.2% vs. 17.5%, throat 4.8% vs. 13.4%, axilla 1.2% vs. 9.1%, and perineum 1.8% vs. 9.5%, respectively. Nares cultures alone detected only 81.5% and 72.5% of all colonized patients by direct and broth-enriched cultures, respectively. The molecular typing of 68 isolates from 17 patients revealed that multisite isolates were largely indistinguishable within each patient, but four patients had multiple subtypes and another three patients had different clonotypes. The detection of MRSA carriers was considerably enhanced by broth-enrichment cultures at multiple anatomic sites and simultaneous colonization by multiple strains at different sites can occur. Epidemiological studies are needed to determine the likelihood of subsequent nosocomial infection among colonized patients detected via direct nasal versus broth-enriched cultures from multiple sites.

Colonisation of fluoroquinolone-resistant Haemophilus influenzae among nursing home residents in southern Taiwan.

Fluoroquinolones have been widely used to treat respiratory tract infections, but fluoroquinolone resistance in Haemophilus influenzae has remained rare. In 2007, prospective surveillance cultures of throat swabs and sputum were conducted every two months on 150 residents of four nursing homes in southern Taiwan. Forty-eight H. influenzae isolates were obtained from 30 (20%) residents. All isolates were non-b serotype and 27 (56.3%) possessed beta-lactamases. Resistance to levofloxacin [minimum inhibitory concentration (MIC) >2 microg/mL] and moxifloxacin (MIC >1 microg/mL) was found in 20 (41.7%) and 21 (43.8%) isolates, respectively. High level levofloxacin and moxifloxacin resistance (MIC >32 microg/mL) was detected in 19 (39.6%) and 15 (31.3%) isolates, respectively. Among 150 residents, those with urinary catheterisation (P=0.018) and tracheostomy tubes (P=0.029) were independently associated with airway colonisation by moxifloxacin-resistant H. influenzae. Among 30 residents with carriage of H. influenzae, no factor was significantly associated with moxifloxacin resistance. Pulsed-field gel electrophoresis of the isolates revealed 14 distinct types. Two major clones accounted for 29 isolates, 27 of which were obtained from 13 residents in one nursing home. All but two of the fluoroquinolone-resistant isolates belonged to these two major clones. This study highlights the emergence of fluoroquinolone-resistant H. influenzae and its clonal spread among nursing home residents in southern Taiwan. Further studies on clinical implications and the extent of fluoroquinolone non-susceptibility and resistance are needed.

Molecular epidemiology of long-term colonization of Candida albicans strains from HIV-infected patients.

Twenty-one Candida albicans isolates from three HIV-infected patients were collected over a period of 3 years and characterized for fluconazole susceptibility, infectivity and genetic relatedness. Fluconazole resistance was found in five isolates, four exhibited dose-dependent susceptibility and the remainder were fully susceptible to this agent. Pulsed-field gel electrophoresis of SfiI restriction digests of the genomic DNA from the isolates revealed that isolates from the same swab specimen were identical despite differences in susceptibility to fluconazole and isolates recovered over time from the three patients retained clonally related DNA fingerprints within each patient. This small-scale study confirms the persistence of oral colonization of C. albicans strains in HIV-infected patients. Clinical data also suggests that the primary infecting strain may become a persistent colonist in the oral cavity once the immune function of the patient has been restored.

Emergence of reduced susceptibility and resistance to fluoroquinolones in Escherichia coli in Taiwan and contributions of distinct selective pressures.

A survey of 1,203 Escherichia coli isolates from 44 hospitals in Taiwan revealed that 136 (11.3%) isolates were resistant to fluoroquinolones and that another 261 (21.7%) isolates had reduced susceptibility. Resistance was more common in isolates responsible for hospital-acquired (mostly in intensive care units) infections (17.5%) than in other adult inpatient (11.4%; P = 0.08) and outpatient isolates (11.9%; P > 0.1). Similarly, reduced susceptibility was more common in isolates responsible for hospital-acquired infections (30.9%) than in other adult inpatient (21.0%; P = 0.04) and outpatient (21.4%; P = 0.06) isolates. Isolates from pediatric patients were less likely to be resistant (1.3 versus 12.0%; P < 0.01) but were nearly as likely to have reduced susceptibility (17.7 versus 21.9%; P > 0.1) as nonpediatric isolates. There was an inverse relationship in the proportion of isolates that were resistant versus the proportion that had reduced susceptibility among isolates from individual hospitals (R = 0.031; P < 0.05). In an analysis of isolates from two hospitals, all 9 resistant strains possessed double point mutations in gyrA and all 19 strains with reduced susceptibility strains had single point mutations; no mutations were found among fully susceptible strains. Risk factors for resistance included underlying cancer (odds ratio [OR], 83; 95% confidence interval [CI(95)], 7.3 to 2,241; P < 0.001), exposure to a quinolone (OR, undefined; P = 0.02), and exposure to a nonquinolone antibiotic (OR, 20; CI(95), 2.2 to 482; P < 0.001); underlying cancer was the only independent risk factor (OR, 83; CI(95), 8.6 to 807; P < 0.001). There were no significant associations between any of these factors and reduced susceptibility. Whereas acute and chronic quinolone use in cancer patients is a major selective pressure for resistance, other undetermined but distinct selective pressures appear to be more responsible for reduced susceptibility to fluoroquinolones in E. coli.

The use of antibiotics critical to human medicine in food-producing animals in Taiwan.

There are increasing public health concerns about antibiotics used in food-producing animals that may contribute to the development of resistance in human pathogens. Such resistance may be critical to human medicine when resistance develops to drugs that treat certain pathogens of which there is no good alternative therapy. We surveyed 10 farms, eight feed mills, and one animal drug distributor in Taiwan to determine the major antibiotics used in food-producing animals, and the extent of use of five drugs that may select for resistance to antibiotics that are critical for human medicine. The five animal drugs, and the resistance of human drug/class they may select for, included avoparcin (vancomycin/glycopeptides), avilomycin (ziracin/envirninomycins), enrofloxacin (ciprofloxacin/fluoroquinolones), virginiamycin (quinupristin and dalfopristin combination/streptogramins), and kanamycin (gentamicin/aminoglycosides). Tetracyclines were the class of antibiotic that was most widely used in the greatest amounts. Over the past 12 months, the number of farms, chicken feed mills, and pig feed mills, that have respectively reported the use of avoparcin was 1 (10%), 5 (63%), 0; avilomycin 0, 0, 3 (50%); enrofloxacin 4 (40%), 1 (13%), 3 (50%); virginiamycin 2 (20%), 5 (63%), 0; and kanamycin 3 (30%), 1 (13%), 1 (17%). We conclude that although the most commonly used antibiotics (ie tetracyclines) have little effect on human medicine, there is a widespread use of antibiotics that may select for critical forms of resistance in human pathogens in food-producing animals.

Outer membrane protein change combined with co-existing TEM-1 and SHV-1 beta-lactamases lead to false identification of ESBL-producing Klebsiella pneumoniae.

Nine isolates of Klebsiella pneumoniae, obtained from one colonized and eight bacteraemic patients on a paediatric ward, were shown to be identical by PFGE, indicating an outbreak. Screening for extended-spectrum beta-lactamase (ESBL) production using the double-disc synergy test, Etest for ESBLs and agar diffusion tests indicated ESBL production. The isolates showed reduced susceptibility to cefotaxime but not to other third-generation cephalosporins. Molecular studies revealed production of TEM-1 and SHV-1 but no ESBLs were identified. Deficiency in expression of an outer membrane protein (OmpK35) was also observed. These observations led us to postulate that the extremely low level of OmpK35 expression and the co-existence of TEM-1 and SHV-1 resulted in an increased MIC of cefotaxime and the false designation of the isolates as ESBL producers. All the infected infants were treated with either third-generation cephalosporins alone or multiple antibiotics including a third-generation cephalosporin, and recovered and were discharged without sequelae.

Comparison of the PACE 2 assay, two amplification assays, and Clearview EIA for detection of Chlamydia trachomatis in female endocervical and urine specimens.

Screening for sexually transmitted diseases (STDs) in a greater proportion of sexually active patients has become an accepted protocol by most health care providers. The purpose of this study was to compare the current test methods for detection of Chlamydia trachomatis used at the University of South Alabama, the PACE 2 assay (Gen-Probe) and the Clearview EIA (Wampole Laboratories), with two amplification technologies, the AMP CT (Gen-Probe) and LCx (Abbott) assays. In addition, a number of demographic parameters were ascertained by asking questions at the time of examination as well as for health care provider concerns and preferences. One urine and four endocervical swab specimens were collected in random order from 787 female patients attending one of four obstetrics-gynecology clinics. Eighty-seven percent of patients had no STD-related symptoms. Patients were considered positive for C. trachomatis if three or more assays (swab and/or urine) were positive. Abbott and Gen-Probe confirmed discrepant results by alternate amplified assays. A total of 66 true-positive specimens were detected by use of the combination of endocervical swabs and urine specimens. After discrepant analysis, sensitivities for endocervical swab specimens for the EIA and the PACE 2, LCx, and AMP CT assays were 50, 81, 97, and 100%, respectively. Sensitivities for the LCx and AMP CT assays with urine specimens were 98 and 81%, respectively. The prevalence of C. trachomatis was 8.4%, as determined by amplification technology. Overall, the amplification technologies were the most sensitive methods with either swab (AMP CT assay) or urine (LCx assay) specimens. The PACE 2 assay offered the advantage of a simpler and less expensive assay with acceptable sensitivity. The clearview CT EIA, while yielding a rapid in-office result, had unacceptably low sensitivity. The wide variation in performance with amplification assays with urine specimens as reported in both this study and the literature obviates the need to clarify optimal parameters for this specimen type.

Surveillance of antibiotic resistance in Taiwan, 1998.

For the first national surveillance of antibiotic resistance in Taiwan, we collected in 1998 from 22 hospitals (6 medical centers, 15 regional hospitals, and 1 local hospital) 3,211 isolates in all parts of the country. Besides 50 random successive isolates from inpatients, each hospital was requested to collect 25 isolates each from positive blood cultures, hospital-acquired infections, outpatients and the pediatric department. We re-speciated all the submitted specimens and determined their antibiotic susceptibility patterns. The most common isolates were Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae), Staphylococcus aureus, and Pseudomonas aeruginosa. Among hospital-acquired infections, Acinetobacter spp. were among those which accounted for over 10% of the isolates. The oxacillin resistance of S. aureus was 82% in isolates from hospital-acquired infections, and 40% from outpatients. Among Enterococcus spp., 85% were either E. faecalis or E. faecium. They were 14% resistant to vancomycin. Among gram-negative bacteria, K. pneumoniae and Acinetobacter baumanii were hospital-acquired isolates that were most clearly more resistant than community acquired isolates. This difference was less apparent in the case of Enterobacter cloacae, Serratia marcescens, and P. aeruginosa. These bacteria were generally more resistant from all sources. Fifty-one percent of Salmonella were resistant to ampicillin; however, these were all sensitive to ciprofloxacin. Isolates from the East were least resistant. Plotting the disc zone diameters of antibiotics within the susceptible range, we identified subpopulations with smaller diameters in the case of vancomycin against S. aureus, ciprofloxacin against E. coli, and ciprofloxacin against Salmonella spp. These findings represent one of the purposes of this surveillance as they may portend developing resistances which bear careful watching in the future.

Evaluation of a rapid air thermal cycler for detection of Mycobacterium tuberculosis.

The Air Thermal Cycler (ATC) (Idaho Technology, Idaho Falls, Idaho) utilizes the unique technology of small-volume glass capillary tubes and high-velocity air for the heating and cooling medium for the PCR. Standard heat block thermal cycler (HBTC) and ATC performance characteristics were compared for the detection of Mycobacterium tuberculosis. Sensitivity was 100% for all smear-positive, M. tuberculosis culture-positive specimens for both the HBTC and the ATC. Of smear-negative, M. tuberculosis culture-positive specimens, sensitivity was 42.9% with the HBTC and 22.0% with the ATC. Specificity was 100% for both assay systems. Total assay time was 6.5 and 4 h and the reagent cost was 84 and 32 cents for the HBTC and ATC, respectively. The ATC offered an excellent alternative to the traditional HBTC for diagnosis of M. tuberculosis in smear-positive specimens by PCR.

Comparison of Bactec 9240 and Difco ESP blood culture systems for detection of organisms from vials whose entry was delayed.

A comparison of the Bactec 9240 (Becton-Dickinson, Sparks, Md.) and Difco ESP (Difco Laboratories, Detroit, Mich.) instruments for the detection of organism growth from vials whose entry was delayed was evaluated. The instruments' capabilities for organism recovery, time to detection, rates of false-positive results, and numbers of vials in which growth was not detected were made by using seeded blood culture vial pairs and controls with and without delayed entry. Bactec 9240 and Difco ESP aerobic and anaerobic vials were inoculated with human blood and were seeded with organism growth from 18 species, including obligate aerobic, anaerobic, and facultative anaerobic organisms. Each organism was tested in triplicate at 0, 8, 24, 36, and 48 h and was incubated at both room temperature (RT) and 35 degrees C. Two separate phases of the study were performed, each with a different version of Bactec 9240 software. Overall, detection of growth in vials with delayed entry into either the Bactec 9240 or the Difco ESP instrument resulted in an increased total time to detection with incubation at both RT and 35 degrees C compared with the total time to detection for nondelayed vials. However, false-positive results and vials in which growth was not detected were minimal, and delayed entry did not require routine entry or exit subcultures for either system. Analysis of individual time points and incubation temperatures for the detection of all organisms suggested that Difco ESP vials delayed by up to 8 h may be incubated at 35 degrees C (100% detection) and vials delayed for longer than 8 h may remain at RT. Bactec 9240 vials may be incubated at 35 degrees C for up to 24 h with a minimal loss of detection (97.9% detection), and vials delayed for more than 24 h should remain at RT for optimal recovery of organism growth.

Hybridization of clinical Escherichia coli isolates from calves and piglets in New York State with gene probes for enterotoxins (STaP, STb, LT), Shiga-like toxins (SLT-1, SLT-II) and adhesion factors (K88, K99, F41, 987P).

Six hundred and sixty-six bovine and fifty-seven swine clinical isolates of E. coli from New York state were examined for the presence of enterotoxins (STaP, STb, LT, SLT-I, and SLT-II) and adhesins (K88, K99, F41, and 987P) using colony hybridization techniques. Three hundred and sixty-seven of the bovine isolates (45.2%) hybridized with at least one gene probe. Of these, two hundred and twenty-three (33.2%) hybridized with F41, one hundred twelve (16.7%) with K99, eighty-two (12.2%) with 987P, ninety-six (14.3%) with STaP, seven (1.1%) with STb, and none (0.0%) with LT and K88. A total of thirty-three (4.7%) of the isolates hybridized with SLT-I, and one (0.1%) with SLT-II. The major pathotypes among the 666 isolates from bovine were K99/F41/StaP (9.8%), K99/F41 (2.5%), p87P/F41 (2.1%) and 987P/K99/F41/StaP (1.4%). Of the swine clinical isolates, twenty-two hybridized with at least one gene probe. The major pathotypes among the isolates from piglets were K88/K99/F41/StaP (5.3%) and K88/F41 (5.3%).

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs.

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli.

Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi. This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD. The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Experimental Lyme disease in dogs produces arthritis and persistent infection.

Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.