Birgit Leonhard

Taurolidine: in vitro Activity against Multiple-Antibiotic-Resistant, Nosocomially Significant Clinical Isolates of Staphylococcus aureus, Enterococcus faecium, and Diverse Enterobacteriaceae

Taurolidine at < or = 1,250 micrograms/ml killed all 37 isolates of multiple-antibiotic-resistant Staphylococcus aureus (n = 9), Enterococcus faecium (n = 17), and Enterobacteriaceae (n = 11). Time-kill experiments disclosed that bovine serum (65% v/v) only marginally retarded the bactericidal activity of 2,000 and 1,000 micrograms/ml of taurolidine against the various strains. Taurolidine at 2,000 micrograms/ml did not antagonize the bactericidal activity of 50% (v/v) fresh human serum against promptly and delayed serum-sensitive test strains of Escherichia coli and Serratia marcescens. In the presence of 65% (v/v) of fresh defibrinated human blood from two donors, however, the bactericidal activity of this antimicrobial compound was delayed, i.e., manifested only following extended (overnight) incubation, against staphylococcal and enterococcal isolates, though less so in the case of Enterobacteriaceae. Taurolidine at 2,000 micrograms/ml killed ingested, i.e., intraphagocytic bacteria of human-serum-resistant S. marcescens strains CDC 06:H3 and P016:H-.

Comparative Susceptibility of Clinical Group A, B, C, F, and G β-Hemolytic Streptococcal Isolates to 24 Antimicrobial Drugs

A total of 312 clinical beta-hemolytic streptococcal isolates (Streptococcus pyogenes, group A = 63; Streptococcus agalactiae, group B = 145; group C = 50; group F = 27; group G = 27) were examined for susceptibility to 23 and 24 antimicrobial drugs with the Bauer-Kirby agar disk diffusion and the agar dilution method, respectively. Sheep blood Mueller-Hinton agar served as the reference medium. Wilkins-Chalgren agar supported optimal growth of group A and B, but not of all group C, F, and G streptococci. The group A streptococci were susceptible to all beta-lactam antibiotics, clindamycin, chloramphenicol, rifampin, teicoplanin, and vancomycin, but resistant to cotrimoxazole, fusidic acid, and, except for 2 strains, to fosfomycin. Resistance (R)/intermediate susceptibility (I) rates (R/I%) to ciprofloxacin (0/2%), ofloxacin (1/2%), erythromycin (1.6/0%), and clarithromycin (0/1%) were low. Higher resistance rates were noted with tetracyclines (doxycycline 23.8/15.9%; tetracycline 39.7/3.2%). Among the group B streptococcal isolates, one strain was resistant against oxacillin and of intermediate susceptibility to penicillin G and cefoxitin. All isolates were susceptible to teicoplanin and rifampin. Conversely, all group B isolates were resistant to cotrimoxazole and fusidic acid; 69% and 51% of these isolates were susceptible to fosfomycin and rifampin, respectively. R/I rates of the group B streptococcal isolates were low for ciprofloxacin and ofloxacin (0/0.7%), clindamycin (0.7/0%), erythromycin (1.4/ 3.5%), clarithromycin (1.4/0%), and chloramphenicol (0.7/0%). Resistance to tetracyclines was significant (doxycycline: 72.4/2.1%; tetracycline; 74.5/1.4%). Among the non-A, non-B beta-hemolytic streptococci, 2 group C strains were resistant to oxacillin and showed intermediate susceptibility to penicillin G. All isolates were susceptible to third and fourth-generation cephalosporins, imipenem, chloramphenicol, rifampin, teicoplanin, and vancomycin. R/I rates to the other antimicrobial drugs were: ciprofloxacin (3.9/1.9%), ofloxacin (2.9/1.9%), clindamycin (2.9/1%), erythromycin (5.8/0%), clarithromycin (3.8/2.9%), and cotrimoxazole (16.4/3.9%). Resistance against tetracyclines was more frequent (doxycycline: 18.3/2.9%; tetracycline: 20.2/6.7%). On the basis of various minor discrepancies between MIC and disk diffusion test results, it is proposed that the current NCCLS inhibition zone (diameter, mm) criteria indicative of intermediate susceptibility of beta-hemolytic streptococci be changed for the following antimicrobial drugs: ampicillin: 22-27 mm (only for group A and B beta-hemolytic streptococci); ciprofloxacin: 16-18 mm; clindamycin: 15-18 mm; doxycycline: 17-19 mm; tetracycline: 17-19 mm, and erythromycin: 14-19 mm.

Surveillance of Pseudomonas aeruginosa in intensive care units: clusters of nosocomial cross-infection and encounter of a multiple-antibiotic resistant strain.

Serogrouping (determination of O antigens) and bacteriocin typing (based on susceptibility to one or more of 18 bacteriocins) were employed to survey 210 isolates of Pseudomonas aeruginosa from 201 patients in 8 intensive care units (ICU) during an observation period of 18 months. Eighty-eight isolates (41.9%) were nonserogroupable (NT); most common were serogroups O1, O9, O11, and O3. All except 5 isolates (97.6%) were bacteriocin-typable. However, phenotypic variation of bacteriocin susceptibility, in particular the receptor for bacteriocin No. 13, rendered this typing method presumptive as well. Bacteriocin susceptibility profiles were not predictive of serogroup and vice versa. Workup of 19 isolates from 9 patients disclosed phenotypic variation of antibiotic susceptibility in 3 patients, superinfection by a different strain in 4 patients, and persistence (3 months) of the same strain in 2 patients, respectively. Serotyping and bacteriocin susceptibility test data revealed 15 clusters of putative cross-infection of 2 patients each, 8 clusters involving 3 patients each, one outbreak (serogroup NT, bacteriocin profile 777736) involving 4 patients in the pediatric ICU, one outbreak due to a multiple-antibiotic resistant (MAR) strain in the surgical ICU (4 patients, serogroup O12, bacteriocin profile 30400), and two putative outbreaks in the pneumonology ICU involving 6 patients (serogroup NT, bacteriocin profile 777726) and 9 patients (serogroup NT, bacteriocin profile 777736). Pulsed-field gel electrophoresis (PFGE) macrorestriction analysis (SpeI, XbaI) confirmed the pediatric and surgical ICU strains as singular strains. However, the two putative outbreaks in the pneumonology ICU were due to one particular strain which had infected 13 of the 15 patients as determined with the PFGE genotypic method. Isolates comprising the MAR strain of P. aeruginosa were susceptible only to amikacin, fosfomycin, and polymyxin B; the isolates varied in susceptibility to aztreonam and ceftazidime. This MAR strain was susceptible to the bactericidal activity of 65 vol% of fresh defibrinated human blood from donors B, L, and T. Either amikacin (16 µg/ml) or fosfomycin (8 µg/ml) plus blood and amikacin (8 µg/ml) combined with fosfomycin (8 µg/ml) with and without blood consistently killed isolates of the MAR strain, which thus was amenable to antibiotic therapy.

Antibiotic susceptibility of Stenotrophomonas (Xanthomonas) maltophilia: comparative (NCCLS criteria) evaluation of antimicrobial drugs with the agar dilution and the agar disk diffusion (Bauer-Kirby) tests.

Ninety-six clinical isolates of Stenotrophomonas maltophilia were examined with the agar dilution method for susceptibility to 19 antimicrobial drugs. Doxycycline, cotrimoxazole, timentin, ofloxacin, fosfomycin, and piperacillin + tazobactam, in that order, inhibited the majority of strains. All isolates were resistant to nitrofurantoin. Concurrent disk susceptibility (Bauer-Kirby method) testing, using currently valid NCCLS interpretative criteria for Pseudomonas aeruginosa, uncovered a significant incidence of very major (category I), major (category II), and minor (categories III and IV) discrepancies for aminoglycosides, cephalosporins, chloramphenicol, and piperacillin + tazobactam and ticarcillin + clavulanic acid. Therefore, new interpretative criteria indicative of intermediate (I) susceptibility of S. maltophilia to these various antibiotics were proposed. In addition, new intermediate susceptibility criteria were proposed for the two β-lactam-β-lactamase inhibitor combinations. It was recommended to exclude ciprofloxacin from test batteries against this microorganism due to the wide scatter of minimal inhibitory concentration values and diameters of inhibition zones; the same was true for polymyxin B. It is hoped that the proposed modified, species- specific criteria will improve the clinical utility of laboratory-generated disk antibiograms with respect to the inherently multiple antibiotic-resistant, opportunistic pathogen S. maltophilia.

Antibiotic Susceptibility Testing (Agar Disk Diffusion and Agar Dilution) of Clinical Isolates of Enterococcus faecalis and E. faecium: Comparison of Mueller-Hinton, Iso-Sensitest, and Wilkins-Chalgren Agar Media

Forty-two isolates of Enterococcus faecalis and 56 isolates of Enterococcus faecium, including 8 vancomycin-resistant strains, were examined for comparative susceptibility to 27 antimicrobial drugs with the agar dilution method, employing Mueller-Hinton (MHA), Iso-Sensitest (ISTA), and Wilkins-Chalgren (WCA) agar. The Bauer-Kirby agar disk diffusion method was used to comparatively test 24 of the agents in parallel. The enterococci yielded better growth on ISTA and WCA. However, WCA completely antagonized co-trimoxazole and, though less, fosfomycin. Importantly, WCA slightly reduced the activities of teicoplanin (minimal inhibitory concentrations, MICs, raised up to twofold) and vancomycin (MICs raised two- to fourfold) against enterococci and staphylococcal quality control strains. Therefore, WCA was judged unsuitable for susceptibility testing of enterococci. For E. faecalis no discrepancies between agar dilution MICs and inhibition zone diameters were encountered with augmentin, ampicillin, ampicillin-sulbactam, chloramphenicol, mupirocin, oxacillin, teicoplanin, and co-trimoxazole. Overall, MHA yielded fewer very major (category I) and major (category II) discrepancies than ISTA. However, numerous minor (category III), slight (category IV), minimal (category V), and/or negligible (category VI) discrepanices were encountered with ciprofloxacin, doxycycline, erythromycin, fosfomycin, fusidic acid, meropenem, ofloxacin and rifampin. With respect to E. faecium, only cefotaxime, mupirocin, oxacillin, and teicoplanin yielded nondiscrepant results. Several very major (I) and major (II) discrepancies were observed with augmentin, ampicillin, ampicillin-sulbactam, doxycycline, fusidic acid, imipenem, and penicillin G. Minor discrepancies (categories III–VI) were particularly numerous with augmentin, chloramphenicol, ciprofloxacin, doxycycline, and piperacillin. The largest numbers of negligible (VI) discrepancies were noted with fosfomycin, fusidic acid, and ofloxacin. It is recommended to test one cephalosporin (cefuroxime or the like) in parallel for educational purposes and to exclude fosfomycin, fusidic acid, and rifampin from test batteries because of the wide scatter of test results. The large number of minimal (V) discrepancies of ciprofloxacin against E. faecalis, the numerous minor (III) and slight (IV) discrepancies of chloramphenicol against E. faecium, and the not insignificant number of very major (I) and minor (III) discrepancies observed with meropenem against isolates of E. faecalis necessitated proposals for new disk intermediate susceptibility criteria.

Antibiotic Susceptibility Testing (Agar Disk Diffusion and Agar Dilution) of Clinical Isolates of Corynebacterium jeikeium

Thirty-three clinical isolates of Corynebacterium jeikeium were examined for susceptibility to 27 antimicrobial drugs with the agar dilution test. Sheep-blood-supplemented Mueller-Hinton agar performed better than Wilkins-Chalgren agar. Disk susceptibility (Bauer-Kirby) tests were carried out in parallel with 24 of the chemotherapeutic agents. All isolates were susceptible to teicoplanin and vancomycin. All isolates resisted fosfomycin, mupirocin, and trimethoprim-sulfamethoxazole. The isolates varied in susceptibility to ciprofloxacin, doxycycline, fusidic acid, ofloxacin, and tetracycline; most were susceptible to rifampin. Surprisingly few discrepancies between agar dilution and disk diffusion tests were encountered when utilizing NCCLS interpretive criteria currently valid for enterococcal isolates.

Stenotrophomonas (Xanthomonas) maltophilia: In vitro Susceptibility to Selected Antimicrobial Drugs, Single and Combined, with and without Defibrinated Human Blood

Sixteen selected isolates of Stenotrophomonas maltophilia varied in susceptibility to the combined phagocytic/serum bactericidal activity of fresh defibrinated human blood (65 vol%). Four representative isolates (X1, X11, X25, and X50), which differed in susceptibility to cefepime, ceftazidime, rifampin, and timentin, were subjected to checkerboard microtiter broth dilution tests involving combinations of cefepime plus timentin, ceftazidime plus ofloxacin, cotrimoxazole plus timentin, rifampin plus polymyxin B, and rifampin plus polymyxin B nonapeptide; all combinations yielded additive or synergistic effects against all four strains. Unexpectedly, the combination of cefepime plus timentin was bactericidally active against the two cefepime-resistant isolates. This finding was substantiated by blood/broth plus combined antimicrobial drug assays. Cefepime plus timentin effectively killed all four test strains. Ofloxacin combined with ceftazidime was bactericidally active against the test strains, including two isolates (X11, X50) with intermediate ofloxacin sensitivity. Cotrimoxazole plus timentin in blood, but not in broth, was bactericidal for the timentin-resistant isolate X25. As expected, various triple combinations of chemotherapeutic agents in blood and broth revealed polymyxin B plus rifampin, regardless of the third combination partner, to exert bactericidal activity against all test strains. Similarly, rifampin combined with ofloxacin and ceftazidime was bactericidally active in blood and broth. The observation that timentin combined with cefepime was effective against cefepime-resistant strains of S. maltophilia might prove of clinical relevance with regard to chemotherapy of nosocomial infections due to multiple-antibiotic resistant strains of this opportunistic pathogen.

Clusters of nosocomial cross-infection due to Acinetobacter baumannii and genospecies 3: Comparison of serotyping with macrorestriction analysis of genomic DNA with pulsed-field gel Electrophoresis

Triplets of isolates representing 20 putative clusters of nosocomial cross-infection due to Acinetobacter baumannii and genospecies 3 were examined comparatively using serotyping and analysis of restriction fragments (SmaI and ApaI) of genomic DNA with the aid of pulsed-field gel electrophoresis. Carbon source assimilation tests disclosed phenotypic variation among 6 to 20 triplets of isolates. Two misleading results of serotyping were encountered. With respect to the presumptive cluster No. 9, one of the genospecies 3 (originally serovar 4) isolates proved to be polyagglutinable upon repeat examination; this particular putative cluster was shown to be a pseudocluster by comparison of the macrorestriction profiles of the respective triple isolates. A strain of A. baumannii serovar 15 had infected 8 patients in a surgical intensive care unit, while a second, genotypically totally different strain of identical serovar had caused infection in one additional patient. With this exception, the correlation between serotyping and analysis of macrorestriction profiles was excellent.

Enterococcus faecium: In vitro Activity of Antimicrobial Drugs, Singly and Combined, with and without Defibrinated Human Blood, against Multiple-Antibiotic-Resistant Strains

The minimal inhibitory (MICs) and bactericidal concentrations of 14 antimicrobial drugs were determined against 17 clinical isolates of Enterococcus faecium, including 4 glycopeptide-resistant strains. Both teicoplanin and vancomycin lacked bactericidal activity against all 13 susceptible isolates. Time-kill experiments served to test various antibiotic combinations chiefly against glycopeptide-resistant strains in Mueller-Hinton broth (MHB) and in MHB supplemented with 65% (v/v) fresh defibrinated human blood. Co-trimoxazole, fusidic acid, and novobiocin yielded bacteriostatic effects. Rifampin was bactericidally active against rifampin-susceptible strains (MICs = 0.125 micrograms/ml), but less so against low-level-rifampin-resistant (MICs = 2-8 micrograms/ml) strains in MHB. However, in the presence of human blood, rifampin (2 micrograms/ml) combined with co-trimoxazole (0.25/4.75 micrograms/ml) killed rifampin-susceptible and low-level-rifampin-resistant, but not moderate-level-rifampin-resistant (MICs = 16-32 micrograms/ml) strains of E. faecium. Of two topical drugs examined, mupirocin merely inhibited strains of E. faecium; conversely, taurolidine at 2,000 micrograms/ml was efficacious against all strains examined, although the kinetics of bactericidal activity were retarded somewhat in the presence of 65 vol% human blood.

Susceptibility of Moraxella catarrhalis to 21 Antimicrobial Drugs: Validity of Current NCCLS Criteria for the Interpretation of Agar Disk Diffusion Antibiograms

Ninety-four clinical isolates of Moraxella catarrhalis were examined for susceptibility to 21 antimicrobial drugs; 67 isolates (= 71.3%) produced beta-lactamase(s). In terms of antibiotic resistance, the number of isolates resistant to penicillin G, ampicillin, and cotrimoxazole were 56, 32, and 1, respectively. The number of isolates with intermediate susceptibility to penicillin G, ampicillin, ciprofloxacin, ofloxacin, cotrimoxazole, and fosfomycin were 11, 34, 1, 2, 2, and 47, respectively. All 94 isolates proved susceptible to ampicillin + 10 micrograms/ml of sulbactam, amoxicillin + 4 micrograms/ml of clavulanic acid, cefuroxime, cefotaxime, cefepime, cefepime, cefixime, imipenem, meropenem, chloramphenicol, doxycycline, tetracycline, fusidic acid, erythromycin, clarithromycin, and rifampin, as based on currently valid NCCLS criteria, where applicable. There were no very major or major discrepancies between agar dilution and agar disk diffusion test results. There were only a few minor discrepancies between test results, specifically: penicillin G (category IV = 4, category VI = 1); ampicillin (category IV = 4, category V = 1, category VI = 7), amoxicillin + clavulanic acid (category III = 11), cotrimoxazole (category IV = 1, category V = 1, category VI = 1), ciprofloxacin (category V = 1), and ofloxacin (category VI = 2). The sole exception was fosfomycin, with a total of 25 minor discrepancies encountered (category III = 14, category V = 9, category VI = 2). Wilkins-Chalgren agar compared favorably with Mueller-Hinton agar following examination with 11 selected antimicrobial drugs against 31 representative isolates of M. catarrhalis.