Risk factors and scoring system for predicting bacterial resistance to cefepime as used empirically in haematology wards.

OBJECTIVES: Bacterial resistance is of growing concern in haematology wards. As the inappropriate administration of empirical antibacterial may alter survival, we studied risk factors for resistance to our usual empirical first-line antibacterial therapy, cefepime. METHODS: We retrospectively studied 103 first episodes of bacteraemia recorded in our haematology department over 2.5 years. Risk factors for cefepime-resistance were identified by multivariate logistic regression with backward selection (P < 0.05). A scoring system for predicting cefepime-resistance was built on independent factor, with an internal validation by the bootstrap resampling technique. RESULTS: 38 (37%) episodes were due to Gram-negative bacteria. Fifty (49%) were due to bacteria resistant to cefepime. Cefepime resistance was significantly associated with a decreased survival at day 30 (P < 0.05). Three risk factors were independently associated with cefepime-resistance: acute lymphoblastic leukaemia; ≥18 days since hospital admission; and receipt of any ß-lactam in the last month. Patients with ≥2 of these risk factors had a probability of 86% (CI 95%, 25 to 100%) to carry a cefepime-resistant strain. CONCLUSION: Using our scoring system should reduce the indication of very broad antibacterial regimens in the empirical, first-line treatment of febrile hematology patients in more than 80% of the cases.

Description and microbiology of endotracheal tube biofilm in mechanically ventilated subjects.

BACKGROUND: A biofilm is found on the inner side of endotracheal tubes (ETT) in mechanically ventilated patients, but its features and role in pneumonia remain unclear. METHODS: This prospective, observational, monocentric study included critically ill ventilated subjects. Measurement of the ETT inner volume was first performed before extubation using the acoustic reflection method. After extubation, the biofilm was studied by means of optical and atomic force microscopy. Bacteriological analysis was then performed and compared with clinical documentation. RESULTS: Twenty-four subjects were included. Duration of intubation lasted from 2 to 79 d (mean ± SD: 11 ± 15 d). The mean percentage of ETT volume loss evaluated in situ (n = 21) was 7.1% and was not linked with the duration of intubation. Analyses with atomic force microscopy (n = 6) showed a full coverage of the inner part of the tube with biofilm, even after saline rinse. Its thickness ranged from 0.8 to 5 µm. Bacteriological cultures of the biofilm (n = 22) often showed the same bacteria as in tracheal secretions, especially for pathogenic organisms. Pseudomonas aeruginosa and Candida albicans were among the most frequent microorganisms. In subjects who had experienced a successfully treated episode of ventilator-associated pneumonia (n = 5), the responsible bacteria were still present in the biofilm. CONCLUSIONS: ETT biofilm is always present in intubated patients whatever the duration of intubation and appears quickly after intubation. Even after soft rinse, a small but measurable part of biofilm remains always present, and seems strongly adherent to the ETT lumen. It contains potentially pathogenic bacteria for the lung.

New procedure to reduce the time and cost of broncho-pulmonary specimen management using the Previ Isola® automated inoculation system.

The microbiological diagnosis of respiratory tract infections requires serial manual dilutions of the clinical specimen before agar plate inoculation, disrupting the workflow in bacteriology clinical laboratories. Automated plating instrument systems have been designed to increase the speed, reproducibility and safety of this inoculating step; nevertheless, data concerning respiratory specimens are lacking. We tested a specific procedure that uses the Previ Isola® (bioMérieux, Craponne, France) to inoculate with broncho-pulmonary specimens (BPS). A total of 350 BPS from a university-affiliated hospital were managed in parallel using the manual reference and the automated methods (expectoration: 75; broncho-alveolar lavage: 68; tracheal aspiration: 17; protected distal sample: 190). A specific enumeration reading grid, a pre-liquefaction step and a fluidity test, performed before the inoculation, were designed for the automated method. The qualitative (i.e., the number of specimens yielding a bacterial count greater than the clinical threshold) and quantitative (i.e., the discrepancy within a 0.5 log value) concordances were 100% and 98.2%, respectively. The slimmest subgroup of expectorations could not be managed by the automated method (8%, 6/75). The technical time and cost savings (i.e., number of consumed plates) reached 50%. Additional studies are required for specific populations, such as cystic fibrosis specimens and associated bacterial variants. An automated decapper should be implemented to increase the biosafety of the process. The PREVI Isola® adapted procedure is a time- and cost-saving method for broncho-pulmonary specimen processing.

Protracted outbreak of multidrug-resistant Acinetobacter baumannii after intercontinental transfer of colonized patients.

OBJECTIVE: To describe the course and management of a protracted outbreak after intercontinental transfer of 2 patients colonized with multidrug-resistant Acinetobacter baumannii (MDRAB). DESIGN: An 18-month outbreak investigation. SETTING: An 860-bed university hospital in France. PATIENTS: Case patients (ie, carriers) were those colonized or infected with an MDRAB isolate. METHODS: During the epidemic period, all intensive care unit (ICU) patients and contacts of carriers who were transferred to wards were screened for MDRAB carriage. Contact precautions, environmental screening, and auditing of healthcare worker (HCW) practices were implemented; rooms were cleaned with hydrogen peroxide mist disinfection. One ICU, in which most of the cases occurred, was closed on 4 occasions for thorough cleaning and disinfection. RESULTS: The 2 index case patients were identified as 2 patients who carried the same MDRAB strain and who were admitted to the hospital after repatriation from Tahiti 5 months apart. During an 18-month period, a total of 84 secondary cases occurred. Reintroduction of MDRAB into the ICUs occurred from patients previously colonized or from healthcare personnel. Termination of the outbreak was only achieved when all carriers from wards or the ICU were cohorted to an isolation unit with dedicated healthcare personnel. CONCLUSIONS: Intercontinental transfer of carriers of MDRAB can result in extensive outbreaks and serious disruption of the hospital's organization. Transmission from carriers most likely occurred via the hands of HCWs, poor cleaning protocols, airborne spread, and contaminated water from sink traps. This protracted outbreak was controlled only after implementation of an extensive control program and eventual cohorting of all carriers in an isolation unit with dedicated healthcare personnel.

Clinical impact and risk factors for colonization with extended-spectrum ß-lactamase-producing bacteria in the intensive care unit.

PURPOSE: The changed epidemiology of extended spectrum beta-lactamases (ESBL), the spread to the community and the need for prudent use of carbapenems require updated knowledge of risk factors for colonization with ESBL-producing enterobacteriaceae (ESBL-PE). METHODS: An 8-month prospective study in the medical ICU of an 850-bed general and university-affiliated hospital. RESULTS: Of 610 patients admitted, 531 (87 %) had a rectal swab obtained at admission, showing a 15 % (82 patients) ESBL-PE carriage rate, mostly of E. coli (n = 51, 62 %); ESBL-PE caused 9 (3 %) infections on admission. By multivariable analysis, transfer from another ICU (OR = 2.56 [1, 22]), hospital admission in another country [OR = 5.28 (1.56-17.8)], surgery within the past year [OR = 2.28 (1.34-3.86)], prior neurologic disease [OR = 2.09 (1.1-4.0)], and prior administration of third generation cephalosporin (within 3-12 months before ICU admission) [OR = 3.05 (1.21-7.68)] were independent predictive factors of colonization by ESBL-PE upon ICU admission. Twenty-eight patients (13 % of those staying for more than 5 days) acquired ESBL carriage in ICU, mostly with E. cloacae (n = 13, 46 %) and K. pneumoniae (n = 10, 36 %). In carriers, ESBL-PE caused 10 and 27 % of first and second episodes of ICU-acquired infections, respectively. CONCLUSION: We found a high prevalence of ESBLE-PE colonization on admission to our ICU, even in the subgroup admitted from the community, but few first infections. Identifying risk factors for ESBL-PE colonization may help identifying which patients may warrant empiric ESBL-targeted antimicrobial drug therapy as a means to limit carbapenem use.

Improving antibiotic use in the hospital: Focusing on positive blood cultures is an effective option.

OBJECTIVES: The unsolicited and systematic evaluation of positive blood cultures (pBC) after laboratory report by a single infectious disease specialist (IDS) was evaluated during one year, using a computer-generated alert by the laboratory. The main objectives of IDS counselling were to improve antibiotic use for bloodstream infection (i.e., initiating or modifying therapy) and to stop unjustified therapy for contaminated pBC. METHODS: During the first part of the study (4 months), all pBC in patients from ICUs, medical and surgical wards were analyzed. After an interim analysis, only pBC from medical and surgical wards were evaluated during the second part (8 months). RESULTS: Overall, 1090 episodes of pBC (representing 866 patients) were evaluated and classified as bloodstream infection (65.5%), contamination (29%) or undetermined (5.5%). Forty-three percent of episodes prompted IDS counselling, including initiation (5%), modification (27.5%), withdrawal (3.5%) and diagnosis workup (5%). Restricting the evaluation to medical and surgical wards increased the rate of counselling (61.2% vs. 27.7%, P<0.0001), notably for de-escalating (20% vs. 8%, P<0.0001), initiating (9% vs. 2%, P<0.0001), oral switch (6% vs. 2%, P<0.0001), withdrawing (5% vs. 2%, P=0.002) or reducing the duration of therapy (5% vs. 2%, P=0.002). DISCUSSION AND CONCLUSIONS: In complement to the laboratory report, a computer-generated alert used by the IDS was useful for the management of pBC in hospital. The impact of IDS counselling was more effective when the evaluation was restricted to medical and surgical wards.

In vitro susceptibility of Actinobaculum schaalii to 12 antimicrobial agents and molecular analysis of fluoroquinolone resistance.

OBJECTIVES: To assess the in vitro susceptibility of Actinobaculum schaalii to 12 antimicrobial agents as well as to dissect the genetic basis of fluoroquinolone resistance. METHODS: Forty-eight human clinical isolates of A. schaalii collected in Switzerland and France were studied. Each isolate was identified by 16S rRNA sequencing. MICs of amoxicillin, ceftriaxone, gentamicin, vancomycin, clindamycin, linezolid, ciprofloxacin, levofloxacin, moxifloxacin, co-trimoxazole, nitrofurantoin and metronidazole were determined using the Etest method. Interpretation of results was made according to EUCAST clinical breakpoints. The quinolone-resistance-determining regions (QRDRs) of gyrA and parC genes were also identified and sequence analysis was performed for all 48 strains. RESULTS: All isolates were susceptible to amoxicillin, ceftriaxone, gentamicin, clindamycin (except three), vancomycin, linezolid and nitrofurantoin, whereas 100% and 85% were resistant to ciprofloxacin/metronidazole and co-trimoxazole, respectively. Greater than or equal to 90% of isolates were susceptible to the other tested fluoroquinolones, and only one strain was highly resistant to levofloxacin (MIC ≥32 mg/L) and moxifloxacin (MIC 8 mg/L). All isolates that were susceptible or low-level resistant to levofloxacin/moxifloxacin (n = 47) showed identical GyrA and ParC amino acid QRDR sequences. In contrast, the isolate exhibiting high-level resistance to levofloxacin and moxifloxacin possessed a unique mutation in GyrA, Ala83Val (Escherichia coli numbering), whereas no mutation was present in ParC. CONCLUSIONS: When an infection caused by A. schaalii is suspected, there is a risk of clinical failure by treating with ciprofloxacin or co-trimoxazole, and ß-lactams should be preferred. In addition, acquired resistance to fluoroquinolones more active against Gram-positive bacteria is possible.

Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR.

BACKGROUND: Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR) assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs). METHODS: Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification). RESULTS: Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%. CONCLUSIONS: This reliable technique may offer a rapid (<1.5 h) tool that would help clinicians to initiate an appropriate treatment earlier. Further investigations are needed to assess the clinical benefit of this novel strategy as compared to phenotypic methods.

Aerococcus urinae and Aerococcus sanguinicola, two frequently misidentified uropathogens.

Species belonging to the Aerococcus genus are isolated from the urine and blood of elderly patients suffering from urinary tract infections (UTIs). However, the clinical significance, phenotypic features and antimicrobial susceptibilities of these underestimated and/or misidentified species remain unclear. From March 2006 to November 2008, among 350 non-enterococcal Streptococcaceae species isolated from urinary specimens, 30 (8.6%) Aerococcus spp. strains were recovered. All strains were characterized using a phenotypic approach (API 20 STREP, ID 32 STREP and VITEK 2 systems), 16S rRNA gene sequencing, and susceptibility to antimicrobial agents commonly used in UTIs. The average age of patients was 73 y and most of them presented with a predisposing urological disease (31%) and/or a systemic underlying condition (48%). All isolates were identified to the species level using the molecular tool (Aerococcus urinae, n = 20; Aerococcus sanguinicola, n = 8; Aerococcus viridans, n = 2), whereas the phenotypic methods were frequently unreliable. All aerococcal isolates were susceptible to amoxicillin, vancomycin, and teicoplanin and showed a low-level resistance to gentamicin. Fluoroquinolones, co-trimoxazole, and fosfomycin exhibited a variable activity. Most A. urinae isolates were resistant to co-trimoxazole and susceptible to fosfomycin, whereas all A. sanguinicola isolates were resistant to fosfomycin and susceptible to co-trimoxazole.

Curbing methicillin-resistant Staphylococcus aureus in 38 French hospitals through a 15-year institutional control program.

BACKGROUND: The Assistance Publique-Hôpitaux de Paris (AP-HP) institution administers 38 teaching hospitals (23 acute care and 15 rehabilitation and long-term care hospitals; total, 23 000 beds) scattered across Paris and surrounding suburbs in France. In the late 1980s, the proportion of methicillin resistance among clinical strains of Staphylococcus aureus (MRSA) reached approximately 40% at AP-HP. METHODS: A program aimed at curbing the MRSA burden was launched in 1993, based on passive and active surveillance, barrier precautions, training, and feedback. This program, supported by the strong commitment of the institution, was reinforced in 2001 by a campaign promoting the use of alcohol-based hand-rub solutions. An observational study on MRSA rate was prospectively carried out from 1993 onwards. RESULTS: There was a significant progressive decrease in MRSA burden (-35%) from 1993 to 2007, whether recorded as the proportion (expressed as percentage) of MRSA among S aureus strains (41.0% down to 26.6% overall; 45.3% to 24.2% in blood cultures) or incidence of MRSA cases (0.86 down to 0.56 per 1000 hospital days). The MRSA burden decreased more markedly in intensive care units (-59%) than in surgical (-44%) and medical (-32%) wards. The use of ABHR solutions (in liters per 1000 hospital days) increased steadily from 2 L to 21 L (to 26 L in acute care hospitals and to 10 L in rehabilitation and long-term care hospitals) following the campaign. CONCLUSION: A sustained reduction of MRSA burden can be obtained at the scale of a large hospital institution with high endemic MRSA rates, providing that an intensive program is maintained for a long period.

Bacteremia in Stevens-Johnson syndrome and toxic epidermal necrolysis: epidemiology, risk factors, and predictive value of skin cultures.

Toxic epidermal necrolysis (TEN) is a rare drug-related life-threatening acute condition. Sepsis is the main cause of mortality. Skin colonization on top of impaired barrier function promotes bloodstream infections (BSI). We conducted this study to describe the epidemiology, identify early predictors of BSI, and assess the predictive value for bacteremia of routine skin surface cultures. We retrospectively analyzed the charts of all patients with Stevens-Johnson syndrome (SJS) and TEN hospitalized over an 11-year period. Blood cultures and skin isolates were recovered from the microbiology laboratory database. Early predictors of BSI were identified using a Cox model. Sensitivity, specificity, and negative and positive predictive values of skin cultures for the etiology of BSI were assessed. The study included 179 patients, classified as having SJS (n = 54; 30.2%), SJS/TEN overlap (n = 59; 33.0%), and TEN (n = 66; 36.9%). Forty-eight episodes of BSI occurred, yielding a rate of 15.5/1000 patient days. In hospital mortality was 13.4% (24/179). Overall, 70 pathogens were recovered, mainly Staphylococcus aureus (n = 23/70; 32.8%), Pseudomonas aeruginosa (n = 15/70; 21.4%), and Enterobacteriaceae organisms (n = 17/70; 24.3%). Variables associated with BSI in multivariate analysis included age >40 years (hazard ratio [HR], 2.5; 95% confidence interval [CI], 1.35-4.63), white blood cell count >10,000/mm3 (HR, 1.9; 95% CI, 0.96-3.61), and percentage of detached body surface area >or=30% (HR, 2.5; 95% CI, 1.13-5.47). Skin cultures had an excellent negative predictive value for bacteremia due to S aureus (especially methicillin-resistant strains) and P aeruginosa, but not for those due to Enterobacteriaceae organisms. In contrast, the positive predictive value was low for all pathogens studied.To our knowledge, this is the largest study describing the epidemiology and risk factors of BSI in patients with SJS/TEN. The body surface area involved is the main predictor of BSI. Excellent negative predictive values of skin cultures for S aureus and P aeruginosa bacteremia should help clinicians consider targeted empirical antibiotic choices when appropriate.

Brachyspira pilosicoli bloodstream infections: case report and review of the literature.

Brachyspira pilosicoli is the etiologic agent of human and animal intestinal spirochetosis and is rarely implicated as a cause of bacteremia. Here, we describe the case of a B. pilosicoli spirochetemia in a 53-year-old male patient suffering from cardiogenic shock. This fastidious bacterium was isolated from blood, likely after translocation from the intestinal tract. Blood cultures were positive after 5 days of incubation (one day after the patient's death), highlighting the problem of the recovery of such type of fastidious bacterium. Identification was achieved by molecular methods (16S rRNA sequencing). A review of the English literature found only 8 cases of bacteremia caused by B. pilosicoli, mostly in immunocompromised or critically ill patients. Finally, difficulties in rapid and accurate diagnosis of B. pilosicoli bloodstream infections, in vitro antimicrobial susceptibility of human clinical isolates, and therapeutic options are discussed.

Campylobacter bacteremia: clinical features and factors associated with fatal outcome.

BACKGROUND: Campylobacter bacteremia is uncommon. The influence of underlying conditions and of the impact of antibiotics on infection outcome are not known. METHODS: From January 2000 through December 2004, 183 episodes of Campylobacter bacteremia were identified in 23 hospitals in the Paris, France, area. The medical records were reviewed. Characteristics of bacteremia due to Campylobacter fetus and to other Campylobacter species were compared. Logistic regression analysis was performed to identify risk factors for fatal outcome within 30 days. RESULTS: Most affected patients were elderly or immunocompromised. C. fetus was the most commonly identified species (in 53% of patients). The main underlying conditions were liver disease (39%) and cancer (38%). The main clinical manifestations were diarrhea (33%) and skin infection (16%). Twenty-seven patients (15%) died within 30 days. Compared with patients with bacteremia due to other Campylobacter species, patients with C. fetus bacteremia were older (mean age, 69.5 years vs. 55.6 years; P = .001) and were more likely to have cellulitis (19% vs. 7%; P = .03), endovascular infection (13% vs. 1%; P = .007), or infection associated with a medical device (7% vs. 0%; P = .02). Independent risk factors for death were cancer (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.2-20.8) and asymptomatic infection (OR, 6.7; 95% CI, 1.5-29.4) for C. fetus bacteremia, the absence of prescription of appropriate antibiotics (OR, 12.2; 95% CI, 0.9-157.5), and prescription of third-generation cephalosporins (OR, 10.2; 95% CI, 1.9-53.7) for bacteremia caused by other species. CONCLUSIONS: Campylobacter bacteremia occurs mainly in immunocompromised patients. Clinical features and risk factors of death differ by infection species.

Emergence of a Streptococcus pneumoniae isolate resistant to streptogramins by mutation in ribosomal protein L22 during pristinamycin therapy of pneumococcal pneumonia.

OBJECTIVES: The aim of this study was to characterize the mechanism of resistance to macrolides and streptogramins of a Streptococcus pneumoniae strain isolated from blood cultures in an 80-year-old patient suffering from severe pneumonia unsuccessfully treated with pristinamycin. METHODS: Resistance genes erm(B) and mef(A) were searched for by PCR. Portions of genes for domains V and II of the 23S rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22 (rplV) were amplified by PCR from total genomic DNA and sequenced. RESULTS: Resistance genes erm(B) and mef(A) were not detected. Only mutation in the rplV gene encoding ribosomal protein L22 was detected. The strain contained a six amino acid insertion ((107)KRTAHI(108)) in the C-terminus of the ribosomal protein L22. CONCLUSIONS: This is the first report of emergence of a pneumococcus resistant to streptogramins by mutation in ribosomal protein L22 during treatment with pristinamycin.

Bacterial identification, clinical significance, and antimicrobial susceptibilities of Acinetobacter ursingii and Acinetobacter schindleri, two frequently misidentified opportunistic pathogens.

The species belonging to the Acinetobacter genus are currently reported as opportunistic pathogens in hospitalized patients with underlying predispositions. However, except for the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, the identification of other species is frequently unreliable, especially for Acinetobacter ursingii and Acinetobacter schindleri, newly described in 2001. Thus, the clinical significance, phenotypic features, and antimicrobial susceptibilities of these two misidentified species remain unclear. Of 456 Acinetobacter sp. clinical strains isolated from 2002 to 2005 in Henri Mondor Hospital, 15 isolates (10 A. ursingii and 5 A. schindleri isolates) were studied. They were characterized using a phenotypic approach (API 20 NE and VITEK 2 systems), 16S rRNA gene sequencing, and susceptibility to antimicrobial agents with evaluation of impact in clinical relevance. The two corresponding type strains were also included for comparison. All isolates were identified to the species level using molecular tools, whereas the phenotypic methods remained unreliable due to the absence of these two species in the manufacturers' databases. However, the API 20 NE system appeared to be a reasonably reliable phenotypic alternative for the identification of A. ursingii when the numerical code 0000071 was found. Conversely, no discriminative phenotypic alternative existed for A. schindleri isolates. Concerning antimicrobial susceptibility, A. ursingii strains appeared to be more resistant to antibiotics than A. schindleri strains, which could imply therapeutic consequences. Finally, the prevalence of infections caused by A. ursingii and A. schindleri (representing 9.7% and 4.8% of non-A. calcoaceticus-A. baumannii complex strains, respectively) seems to be underestimated.

Applying the concept of healthcare-associated infections to hematology programs.

Infection is a leading cause of mortality in hematology. Although data on nosocomial infections are available, little is known about events falling into the broader category of healthcare-associated infections. Our aim was to evaluate the incidence and causes of healthcare-associated infections in hematology patients, comparatively with nosocomial infections. Using predefined criteria, we classified 223 infectious episodes in 137 patients for their association with healthcare and nosocomial occurrence. Of the 223 infectious episodes, 204 (91%) were healthcare associated, 94/223 (42%) were also nosocomial, and 9% were community-acquired. Healthcare-associated infections should be preferred to nosocomial infections--which underestimates half of the healthcare-associated infections--as quality indicators for preventive programs.

Association between hand hygiene compliance and methicillin-resistant Staphylococcus aureus prevalence in a French rehabilitation hospital.

We simultaneously investigated the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and compliance with hand hygiene in the clinical wards of a French rehabilitation hospital. We found that the rate of hand hygiene compliance observed at the patient's bedside was a strong predictor of MRSA prevalence.

In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid.

OBJECTIVES: To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis. METHODS: Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype. RESULTS: The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC reproduced the original phenotype in E. coli KL16. CONCLUSIONS: A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.