Wild-type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the Clinical and Laboratory Standards Institute broth microdilution methods.

Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were < or = 2 microg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of < or = 1 microg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of < or = 0.25 microg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10 kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n = 21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (≥9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

The recovery of Mycobacterium avium complex and Mycobacterium tuberculosis from blood specimens of AIDS patients using the nonradiometric BACTEC NR 660 medium.

The ability of the nonradiometric BACTEC NR 660 aerobic 6A blood culture medium to support mycobacterial growth was investigated. During a 19-month period blood cultures from 140 AIDS patients were sent to the microbiology laboratory. After the cultures were incubated for seven days, aliquots of medium from the vials were centrifuged, sediments examined microscopically for mycobacteria, and cultured to mycobacterial media. Seventy-one AIDS patients (51%) had at least one blood culture positive for mycobacteria. There was a significant difference in the percent of female AIDS patients positive for mycobacteria compared to male patients (72% vs. 44%, P less than 0.01). Forty-four percent of all subsequently positive cultures were detected by an acid fast stain of the specimen sediment. Subcultures from the BACTEC 6A suspensions were positive on mycobacterial media at one-seven weeks (mean three weeks) after planting. Sixty-nine of the isolates were Mycobacterium avium complex, while two were Mycobacterium tuberculosis. Some bacteremias with M. tuberculosis may have been undetected because growth experiments with a reference strain showed that, in contrast to M. avium complex, M. tuberculosis did not increase in concentration in 6A medium.

Plasmodium falciparum: comparison of in vitro growth of knobby and knobless isolates.

Variants (K-) of three strains of Plasmodium falciparum which do not produce the erythrocyte surface alterations that have been called knobs have been compared with their wildtype knobby (K+) parents. The K- variants achieve higher parasitemias, incorporate radiolabeled isoleucine more rapidly, and produce a higher percentage of multiply-infected cells than do their K+ parents. Nevertheless, immune owl monkey sera cause approximately the same percentage inhibition of growth of both K+ and K- organisms when included in the growth medium at a 1% concentration.

Reaction of immune sera with components of the human malarial parasite, Plasmodium falciparum.

Human and monkey sera from individuals exposed to Plasmodium falciparum were characterized by indirect immunofluorescence, in vitro parasite growth inhibition, and immunoprecipitation of 125I-labeled parasite antigens followed by analytical sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In general there was a good correlation between fluorescence titer and the ability of a serum to inhibit parasite growth in vitro. Exceptions were found, however. Some variance was seen in the ability of a given serum to inhibit different strains of the parasite. The significance of this is unknown. The proteins bound by human sera with high and low in vitro inhibitory capacities were compared by SDS-PAGE. The human sera which did not inhibit parasite growth in vitro well differed from those which did by failing to efficiently bind certain parasite components having molecular weights in the range of 200,000, 70,000-85,000, and 45,000.

Diagnosis of herpesvirus infections: correlation of Tzanck preparations with viral isolation.

Evaluation of Giemsa-stained smears of scrapings from the base of vesicles or ulcers ( Tzanck preparation) for the presence of multinucleated giant cells and/or intracellular inclusions were diagnostic for herpesvirus in 18 of 21 cases (86%) of culturally proved herpesvirus infections. Smears from four patients with varicella-zoster infection also revealed cytologic alterations characteristic of herpesvirus.

Bowel colonization with resistant gram-negative bacilli after antimicrobial therapy of intra-abdominal infections: observations from two randomized comparative clinical trials of ertapenem therapy.

The selection of resistant gram-negative bacilli by broad-spectrum antibiotic use is a major issue in infection control. The aim of this comparative study was to assess the impact of different antimicrobial regimens commonly used to treat intra-abdominal infections on the susceptibility patterns of gram-negative bowel flora after completion of therapy. In two international randomized open-label trials with laboratory blinding, adults with complicated intra-abdominal infection requiring surgery received piperacillin-tazobactam (OASIS 1) or ceftriaxone/metronidazole (OASIS II) versus ertapenem for 4-14 days. Rectal swabs were obtained at baseline, end of therapy, and 2 weeks post-therapy. Escherichia coli and Klebsiella spp. were tested for production of extended-spectrum beta-lactamase (ESBL). Enterobacteriaceae resistant to the agent used were recovered from 19 of 156 (12.2%) piperacillin-tazobactam recipients at the end of therapy compared to 1 (0.6%) patient at baseline (p<0.001) in OASIS I, and from 33 of 193 (17.1%) ceftriaxone/metronidazole recipients at the end of therapy compared to 5 (2.6%) patients at baseline (p<0.001) in OASIS II. Ertapenem-resistant Enterobacteriaceae were recovered from 1 of 155 and 1 of 196 ertapenem recipients at the end of therapy versus 0 and 1 ertapenem recipients at baseline in OASIS I and II, respectively. Resistant Enterobacteriaceae emerged significantly less often during treatment with ertapenem than with the comparator in both OASIS I (p<0.001) and OASIS II (p<0.001). The prevalence of ESBL-producers increased significantly during therapy in OASIS II among 193 ceftriaxone/metronidazole recipients (from 4 [2.1%] to 18 [9.3%]) (p<0.001), whereas no ertapenem recipient was colonized with an ESBL-producer at the end of therapy in either study. Selection for imipenem-resistant Pseudomonas aeruginosa was uncommon in all treatment groups. In these studies, the frequency of bowel colonization with resistant Enterobacteriaceae substantially increased in patients treated with either piperacillin-tazobactam or ceftriaxone/metronidazole, but not in patients treated with ertapenem.

Inhibition of the in vitro growth of Plasmodium falciparum. I. The effects of immune serum and purified immunoglobulin from owl monkeys.

Sera from Aotus sp. monkeys (karyotypes II, III, and IV) which were immune to Plasmodium falciparum have been used to inhibit the in vitro growth of this human malaria parasite. Culture conditions used for the assays allowed 50- to 100-fold increases in the number of A+ erythrocytes infected in a 96-hr period in control cultures. Although normal monkey serum did not support growth as well as normal human serum, mixtures of normal monkey and human serum were found that did. Compared to such controls, as little as 3.5% immune monkey serum was found to cause approximately 56% inhibition in 4 days (2 replicative cycles). Purified globulin from immune monkeys inhibited 40% at 2 mg/ml and 75% at 7 mg/ml after a single replicative cycle. These data suggest that serum antibody is likely to play a major role in providing Aotus monkeys with protective immunity to P. falciparum.

Cephalothin susceptibility as a potential marker for the Aeromonas sobria group.

Fifty-four motile Aeromonas strains, composing the three currently recognizable species, were tested for susceptibility to cephalothin by broth dilution and disk agar diffusion assays. Cephalothin susceptibility was significantly associated with Aeromonas sobria (P less than 0.001) and may be an additional phenotypic marker useful in the identification of this species.

In vitro susceptibilities of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae to 22 antimicrobial agents.

MICs of 22 antimicrobial agents for 60 strains of three Aeromonas species were determined by a microdilution method. The newer cephalosporins such as moxalactam, cefotaxime, and cefoperazone, the aminoglycosides, and chloramphenicol, tetracycline, nitrofurantoin, and trimethoprim-sulfamethoxazole inhibited most of the strains studied. Within the genus, A. hydrophila was more resistant than either A. caviae or A. sobria to the antibiotics tested.

Rapid detection of positive blood cultures with the BACTEC NR-660 does not require first-day subculturing.

An analysis of blood culture data was performed to determine whether subculturing within the first 24 h of incubation decreased the time to detection of positive blood cultures when compared with the routine use of the BACTEC NR-660 system (Johnston Laboratories, Inc., Towson, Md.). During a 9-month period (June 1985 to February 1986), 17,913 blood cultures were received in our laboratory, of which 1,463 (8.2%) became positive. Of the positive cultures, 97% were detected with equal or greater rapidity by the NR-660 system than by visual inspection and first-day blind subculturing. There were 37 delayed positive cultures from which only one isolate (0.07%) was not eventually detected by the NR-660 system. Coagulase-negative staphylococcus was the most frequent isolate among the delayed positive cultures, but only 3 of 15 isolates were known to be clinically significant isolates. The longest delay in detection by the NR-660 system was 6 days for one isolate of Cryptococcus neoformans and one isolate of Klebsiella pneumoniae. Although subculturing may decrease the time to detection of a few cultures, the majority of positive blood cultures were detected faster or with equal speed by the NR-660 system. When the data were evaluated, routine use of the NR-660 system was sufficient for the detection of positive blood cultures and was cost-effective.