Species Identification and Antifungal Susceptibility Patterns of Species Belonging to Aspergillus Section Nigri

A phylogenetic analysis was performed for 34 Aspergillus strains belonging to section Nigri. Molecular methods allowed for the correct classification into three different clades (A. niger, A. tubingensis, and A. foetidus). Correlation with in vitro itraconazole susceptibility distinguished the following three profiles: susceptible, resistant, and showing a paradoxical effect. A number of different species whose morphological features resemble those of A. niger showed unusual MICs to itraconazole that have never been described for the Aspergillus genus.Black aspergilli are widely distributed in nature (16); they are common food spoilers but are also well used for industrial purposes (15). Among Aspergillus species of the Nigri group, A. niger constitutes the most frequent etiological agent of otomycosis (13) and is considered the third cause of pulmonary aspergillosis (10). Nevertheless, the clinical implications of other species are rarely reported, and they are generally identified as A. niger (14, 22).Clinically, identification of unknown Aspergillus clinical isolates to the species level may be important given that different species have dissimilar susceptibilities to antifungal drugs. Thus, the knowledge of the species identity may influence the choice of appropriate antifungal therapy (2, 4). Furthermore, since the antifungal susceptibility patterns for most of the species within section Nigri have been poorly investigated, their identification and antifungal susceptibility profiles appear to be of clinical interest for further research.Black aspergilli belong to one of the most difficult groups concerning classification and identification (18), and so a number of different techniques have been developed in order to solve this issue. Among them, molecular tools are the gold standard (1, 18), as the sequencing of the β-tubulin or calmodulin gene is suitable, and enough, to discriminate between species within section Nigri (3, 18, 21).Thirty-four Aspergillus section Nigri strains belonging to the Mold Collection of the Centro Nacional de Microbiologia and collected since 2004 were analyzed. Thirty-three strains were independent clinical isolates, and 1 had an environmental origin. All strains were identified as A. niger using conventional methods of morphology at the macroscopic as well as microscopic levels (9). Species identification analysis was addressed using sequences of the β-tubulin gene from all the strains included in this study together with the sequences of different Aspergillus section Nigri type strains and others that were available at GenBank as follows: A. tubingensis {"type":"entrez-nucleotide","attrs":{"text":"AY820007","term_id":"65307061","term_text":"AY820007"}}AY820007^T, {"type":"entrez-nucleotide","attrs":{"text":"AY820009","term_id":"65307063","term_text":"AY820009"}}AY820009, and {"type":"entrez-nucleotide","attrs":{"text":"AY585527","term_id":"53830386","term_text":"AY585527"}}AY585527; A. foetidus {"type":"entrez-nucleotide","attrs":{"text":"AY585533","term_id":"53830397","term_text":"AY585533"}}AY585533^T, {"type":"entrez-nucleotide","attrs":{"text":"AY585534","term_id":"53830399","term_text":"AY585534"}}AY585534, and {"type":"entrez-nucleotide","attrs":{"text":"DQ768454","term_id":"111609935","term_text":"DQ768454"}}DQ768454; and A. niger {"type":"entrez-nucleotide","attrs":{"text":"FJ629288","term_id":"225904264","term_text":"FJ629288"}}FJ629288^T, {"type":"entrez-nucleotide","attrs":{"text":"EF422213","term_id":"126105445","term_text":"EF422213"}}EF422213, and {"type":"entrez-nucleotide","attrs":{"text":"AY585537","term_id":"53830403","term_text":"AY585537"}}AY585537. Partial sequences of the β-tubulin gene were amplified using the primer set βtubAniger1 and βtubANiger2 (11) and were carried out according to standard PCR guidelines (Applied Biosystems). Sequences were assembled and edited using the SeqMan II and EditSeq software packages (Lasergene 8.0; DNAStar, Inc., Madison, WI).All phylogenetic analyses were conducted with InfoQuest FP software, version 4.50 (Bio-Rad). The methodology used was maximum parsimony clustering. Phylogram stability was assessed by using parsimony bootstrapping with 2,000 simulations and by using the Aspergillus clavatus {"type":"entrez-nucleotide","attrs":{"text":"AY214441","term_id":"32441964","term_text":"AY214441"}}AY214441^T sequence as the out-group.The phylogenetic tree grouped the 34 clinical isolates into three different clades consisting of 13 A. niger isolates, 18 A. tubingensis isolates, and 3 A. foetidus isolates (Fig. ​(Fig.1).1). Table ​Table11 shows β-tubulin gene identification, as well as the origin and susceptibility profiles of the isolates.Open in a separate windowFIG. 1.Phylogenetic tree using maximum parsimony phylogenetic analysis and 2,000 bootstrap simulations based on β-tubulin gene sequences from all the Aspergillus section Nigri strains included in the study. Percentages indicate the bootstrap support for each group of sequences. (T), type strain.

TABLE 1.

Source, molecular identification, MICs, and MECs for species of Aspergillus section Nigri^a

Use of Pristinamycin for Infections by Gram-Positive Bacteria: Clinical Experience at an Australian Hospital

Thirty-six patients were treated with pristinamycin for 46 different microbiological isolates between April 2007 and July 2009. Pathogens included 9 methicillin-resistant Staphylococcus aureus isolates, 13 methicillin-resistant coagulase negative staphylococci, and 9 vancomycin-resistant enterococci. Sites of infections included 12 osteomyelitis cases, 10 prosthetic joints, 4 other prostheses, and 1 epidural abscess. Five patients ceased treatment due to side effects. Ten patients were cured of their infections, and 21 patients had infections successfully suppressed.The management of Gram-positive infections is becoming increasingly difficult as multidrug-resistant bacteria become more prevalent (http://www.health.vic.gov.au/__data/assets/pdf_file/0005/331727/vre-report.pdf). Many complex infections require prolonged antibiotic therapy. The current therapeutic options have limitations, such as emerging bacterial resistance, the need for intravenous access, side effects, and cost (10).Pristinamycin is an oral streptogramin antibiotic made up of two synergistic but structurally unrelated components, pristinamycin IA and pristinamycin IIA. Separately, each component has bacteriostatic activity. Together, they have bactericidal action (8). Pristinamycin has a wide range of in vitro activity, including against most Gram-positive cocci and Clostridium, Haemophilus, Neisseria, Chlamydia, and Ureaplasma species (1, 5, 6). Enterococcal species generally are less sensitive to pristinamycin than staphylococci or streptococci in vitro (6). This may be because enterococci are naturally resistant to streptogramin A (4). Most enterococci that also are resistant to streptogramin B remain sensitive to the combination of agents in pristinamycin (2, 6). There have been only limited reports of its use in the English-language literature (3, 7).This retrospective study was carried out at a single adult tertiary hospital in Australia. Pharmacy dispensing records were searched to identify all patients prescribed pristinamycin up to July 2009. One patient was excluded, as only two doses of pristinamycin were administered and no conclusions could be drawn. A single trained researcher viewed the medical records to collect information regarding patient management and outcome. Data were collected up to a predetermined study closure date of 1 September 2009. Cure was defined as no evidence of recurrence at follow-up after the cessation of antibiotics. Suppression was defined as improvement in imaging, inflammatory markers, and clinical condition while continuing antibiotic treatment. Failure was defined as either the lack of a clinical response to treatment or intolerance requiring the withdrawal of treatment. Where polymicrobial infection existed, the outcome was attributed to all organisms susceptible to pristinamycin. The dose and duration of pristinamycin therapy was at the discretion of the prescribing physician. There is no standardized testing method for pristinamycin susceptibility. Testing for in vitro susceptibility to quinupristin-dalfopristin by Vitek 2 was used as a proxy for pristinamycin susceptibility (7, 9). Other antibiotic susceptibilities were determined according to standard NCCLS criteria. Coagulase-negative staphylococci (CNS) were grouped together and classified as either methicillin-sensitive CNS or methicillin-resistant CNS based on in vitro susceptibility testing.Thirty-six patients were treated with pristinamycin for 46 microbiological isolates. Table ​Table11 details the patient characteristics. Bone and joint infection was the most common indication, with 12 patients having osteomyelitis and 10 having prosthetic joint infections. Other indications included one prosthetic mitral valve endocarditis, one permanent pacemaker infection, two prosthetic vascular graft infections, four postoperative wound infections, two intravenous-line-related bacteremias, two intraabdominal infections, one urinary tract infection, and one epidural abscess. Tables ​Tables22 and ​and33 show details of individual patients and outcomes stratified to patient demographics.

TABLE 1.

Patient demographics and comorbidities

Antianaerobic Activity of Sulopenem Compared to Six Other Agents

Agar dilution MIC methodology was used to compare the activity of sulopenem with those of amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 431 anaerobes. Overall, MIC₅₀/₉₀ values were as follows: sulopenem, 0.25/1.0 μg/ml; amoxicillin/clavulanate, 0.5/2.0 μg/ml; ampicillin/sulbactam, 0.5/4.0 μg/ml; piperacillin/tazobactam, 0.25/8.0 μg/ml; imipenem, 0.06/1.0 μg/ml; clindamycin, 0.25/16.0 μg/ml; and metronidazole, 1.0/4.0 μg/ml.Anaerobes are becoming increasingly resistant to β-lactams due to β-lactamase production and other mechanisms. Although β-lactamase production, and concomitant resistance to β-lactams, is the norm among the Bacteroides fragilis group, other anaerobic gram-negative bacilli in the genera Prevotella, Porphyromonas, and Fusobacterium have increasingly become β-lactamase positive. β-Lactamase production also has been described in clostridia. Metronidazole resistance in organisms other than non-spore-forming gram-positive bacilli has been described, as has clindamycin resistance in anaerobic gram-negative bacilli (1-5).Although most anaerobes are susceptible to carbapenems, resistance has occurred. Quinolone resistance has developed, importantly in Clostridium difficile, necessitating development of other agents to treat pseudomembranous colitis (6, 8-10, 12, 14, 16).CP-65,207, whose in vitro activity was published in 1989 (13), is a diastereomeric mixture of two isomers, the active component of the two being sulopenem. Sulopenem development was put on hold in the 1990s as discovery/development efforts were focused on gram-positive organisms. There is now an urgent need for drugs against gram-negative strains, and sulopenem has reentered clinical development (M. Huband, personal communication). PF-03709270 is a novel oral prodrug of sulopenem (11). The oral prodrug approach makes this compound unique among the penems and carbapenems. PF-03709270 and sulopenem both entered phase II studies in December 2008. The structures of sulopenem and its oral prodrug PF-03709270 are shown in Fig. ​Fig.11 and ​and2,2, respectively.Open in a separate windowFIG. 1.Chemical structure of sulopenem.Open in a separate windowFIG. 2.Chemical structure of PF-03709270, the oral prodrug of sulopenem.This study examined the activity of sulopenem compared with those of amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, imipenem, clindamycin, and metronidazole against 431 anaerobes.(This study was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008 [9a].)All anaerobes were clinical strains identified by standard procedures (16) and kept frozen in double-strength skim milk (dehydrated skim milk; BD, Sparks, MD) at −70°C until use. Prior to testing, strains were subcultured twice onto enriched brucella agar plates (7). Sulbactam and sulopenem susceptibility powders were obtained from Pfizer Central Research, Groton, CT, and other drugs were obtained from the respective manufacturers. β-Lactamase testing was performed by the nitrocefin disk method (Cefinase; BD, Sparks, MD). Agar dilution susceptibility testing was performed according to the latest method recommended by the Clinical and Laboratory Standards Institute (CLSI) (7), using brucella agar with 5% sterile laked sheep blood. Clavulanate and sulbactam were combined with amoxicillin and ampicillin, respectively, in 1:2 ratios, and tazobactam with piperacillin at a fixed concentration of 4.0 μg/ml. Breakpoint values, where CLSI approved, were as follows: amoxicillin/clavulanate, ≤4.0 μg/ml; ampicillin/sulbactam, ≤8.0 μg/ml; piperacillin/tazobactam, ≤32.0 μg/ml; imipenem, ≤4.0 μg/ml; clindamycin, ≤2.0 μg/ml; and metronidazole, ≤8.0 μg/ml. Quality control strains recommended by CLSI (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eggerthella lenta ATCC 43055) were included with each run. Sulopenem MIC ranges for these strains were 0.06 to 0.125, 0.25, and 1.0 μg/ml, respectively.Among the anaerobic gram-negative bacilli tested, 95/101 (94%) of B. fragilis group strains, 57/100 (57%) of Prevotella/Porphyromonas strains, and 3/60 (5%) of fusobacterial strains produced β-lactamase. Results of MIC testing are presented in Table ​Table1.1. Overall, MIC₅₀/₉₀ values were as follows: sulopenem, 0.25/1.0 μg/ml; amoxicillin/clavulanate, 0.5/2.0 μg/ml; ampicillin/sulbactam, 0.5/4.0 μg/ml; piperacillin/tazobactam, 0.25/8.0 μg/ml; imipenem, 0.06/1.0 μg/ml; clindamycin, 0.25/16.0 μg/ml; and metronidazole, 1.0/4.0 μg/ml.

TABLE 1.

MICs of the agents tested in this study

In Vitro Sensitivity and Resistance of 46 Leptospira Strains Isolated from Rats in the Philippines to 14 Antimicrobial Agents

The in vitro susceptibilities of 46 Leptospira isolates from rats to 14 antimicrobial agents were tested. All of the strains were found to be sensitive to ampicillin, cefotaxime, ciprofloxacin, norfloxacin, doxycycline, erythromycin, and streptomycin. In contrast, the tested isolates showed resistance to amphotericin B, 5-fluorouracil, fosfomycin, trimethoprim, sulfamethoxazole, neomycin, and vancomycin. These findings will help in selecting effective and ineffective antimicrobials for treatment of leptospirosis and for the development of new selective media, respectively.Pathogenic Leptospira species, spirochetes of the genus Leptospira, are the causative agents of leptospirosis, a zoonotic disease with a worldwide distribution. These bacteria are carried by a variety of animal species in their kidney tubules and contaminate the environment by being shed in urine (2). Contact with this contaminated environment is one of the important pathways of transmission of the disease. Although this disease is associated with remarkable rates of mortality and morbidity in both animals and humans, diagnosis is often difficult due to nonspecific clinical presentation and limitations in laboratory tests. Due to these difficulties, clinically suspected cases are usually treated by empirical chemotherapy, for which information about the susceptibilities of Leptospira isolates against various antimicrobial agents is required. Several studies have been reported previously regarding the susceptibilities of Leptospira isolates to a large number of antimicrobial agents (6, 8, 11, 12, 15, 18, 19). However, most of these reports used laboratory-passaged strains. Studies revealing the sensitivities of clinical and environmental isolates are few (18, 19) and limited by the use of a small number of strains.In comparison with the available data on effective antimicrobials, published data reporting agents that are ineffective against Leptospira strains are rare. For improving the isolation of leptospires from contaminated samples, information regarding ineffective antimicrobials is helpful in the selection of agents to prevent the usual contamination in Leptospira cultures.In the present study, we determined the susceptibilities of 46 Leptospira strains to 14 antimicrobials, including both effective and ineffective agents. To screen for sensitivity to antimicrobials, we performed MIC assays of aminobenzyl penicillin (ampicillin), new quinolone (ciprofloxacin and norfloxacin), tetracycline (doxycycline), aminoglycoside (streptomycin), macrolide (erythromycin), and expanded-spectrum cephalosporin (cefotaxime) for these strains. Among the tested antimicrobials, penicillins, doxycycline, and broad-spectrum cephalosporins are widely used for the treatment of leptospirosis (2, 17, 25). The other drugs were also found to be effective against Leptospira isolates in several in vitro (6, 8, 11, 12, 15, 18, 19) and in vivo (3, 5, 17, 20, 21, 22) studies.To test susceptibility to ineffective agents, we selected 5-fluorouracil, fosfomycin, neomycin, and vancomycin due to their usage in selective media for culture of Leptospira isolates (1, 7, 13, 14, 16, 23). We also included amphotericin B for its antifungal effect and trimethoprim-sulfamethoxazole due to their use in the selective medium of another spirochete, Borrelia (10).The Leptospira strains used in this study were collected from rats in Metro Manila and Laguna, Philippines, from August 2006 to May 2007 (24). During isolation, Ellinghausen-McCullough-Johnson-Harris (EMJH) and Korthof''s media supplemented with 5-fluorouracil were used for primary culture of the rat kidneys. These isolates were classified as Leptospira interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa and Leptospira borgpetersenii serogroup Javanica (see Table S1 in the supplemental material). Of the 46 strains, 34 were lethal in experimental infections of golden hamsters (24). All of these isolates had undergone fewer than 10 in vitro passages before our experiment. Both Korthof''s and EMJH medium (2, 25) were used for subculturing the strains. To compare the validity of our test with the results of previous reports, we used Leptospira interrogans strain Ictero No. 1 (serovar Icterohaemorrhagiae), which was maintained in our laboratory through continuous passages in Korthof''s medium. For preparing the inocula, cultures were grown at 30°C for 7 days and enumerated in a Thoma counting chamber under dark-field microscopy.The antimicrobial agents were purchased from Sigma-Aldrich, except for trimethoprim (Wako Pure Chemical Industries Ltd., Tokyo, Japan). To prepare the stock solutions, antimicrobials were dissolved in sterile distilled water, except that ciprofloxacin and norfloxacin were dissolved in 0.1 N NaOH, erythromycin, sulfamethoxazole, and trimethoprim in 95% ethanol, and amphotericin B in dimethyl sulfoxide. Further dilutions of these agents were done with either sterile distilled water or Leptospira media. The stock solutions were stored at −80°C in one-time-use aliquots.The broth microdilution method was performed as previously described by Murray and Hospenthal (11). The MICs were noted visually as the lowest concentration of antimicrobials in the wells without color change of alamar blue from blue to pink/purple. We repeated this test at least twice for each strain, and the MIC of cefotaxime for strain K56 was confirmed in three repeated tests. The results were reproducible for each antimicrobial-strain combination.Results showing the distribution of MIC values are displayed in two groups: effective and ineffective antimicrobials are shown in Table ​Table11 and in Table ​Table2,2, respectively. The MIC₅₀s and MIC₉₀s of the strains are included in the tables. From our results, all of the strains were found to be sensitive to ampicillin, ciprofloxacin, doxycycline, erythromycin, and streptomycin, which correlates with previous studies (6, 8, 11, 12, 15, 18, 19). Norfloxacin, which has not been tested before for antileptospiral activity, was also found to be effective against these strains (Table ​(Table1).1). As resistance to ciprofloxacin is rapidly emerging among Escherichia coli and other enteric bacteria (4, 9), this finding supports the use of norfloxacin instead of ciprofloxacin as an alternative choice in empirical therapy. In the case of cefotaxime, though most of the strains showed sensitivity, the MICs for a few strains were higher than in the previous reports. This reduced effectiveness of cefotaxime against some strains requires further investigation to survey the development of resistance of Leptospira strains against this potent antileptospiral agent. Regardless of this finding, all of these antimicrobials were found to have potential as chemotherapeutic agents for leptospirosis due to their high efficacies, lower costs, and eligibility for empirical therapy.

TABLE 1.

Distribution of MICs of ampicillin, cefotaxime, ciprofloxacin, norfloxacin, doxycycline, erythromycin, and streptomycin among the Leptospira strains

First Detection of CTX-M and SHV Extended-Spectrum β-Lactamases in Escherichia coli Urinary Tract Isolates from Dogs and Cats in the United States

One hundred fifty canine and feline Escherichia coli isolates associated with urinary tract infections were screened for the presence of extended-spectrum β-lactamase (ESBL) genes. Out of 60 isolates suspected to be ESBL positive based on antimicrobial susceptibility testing, 11 ESBLs were identified, including one SHV-12 gene, one CTX-M-14 gene, and nine CTX-M-15 genes. This study provides the first report of CTX-M- and SHV-type ESBLs in dogs and cats in the United States.The first detection of an extended-spectrum β-lactamase (ESBL) in an organism from an animal was reported in Japan in 1988 in an Escherichia coli isolate from a laboratory dog (13). Since that time, numerous reports of ESBL-positive isolates from dogs and cats, as well as from other animal species (5), have been made worldwide (4, 8, 11, 17, 25, 26). Only one study has identified the presence of an ESBL in isolates from animals in the United States, i.e., in Salmonella enterica serovar Newport from horses (21). We hypothesized that ESBL genes would be present in urinary E. coli isolates from companion animals in the United States. The purpose of this study was to screen a group of 150 E. coli isolates from canine and feline patients that had been diagnosed with a urinary tract infection (UTI) for the presence of ESBL genes.A convenience sample of 150 E. coli isolates collected from canine and feline patients at the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania with clinical signs or evidence on routine urinalysis of a UTI between 1 September 2004 and 31 December 2007 was used in this study. Isolates were frozen in Microbank tubes (ProLab Diagnostics, Austin, TX) and stored at −70°C prior to use.MICs were determined using a Negative Combo 31 panel on a MicroScan Walkaway 40 (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, IL). Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) breakpoints (7). Isolates collected from the same individual animal within a 45-day period were considered to be the same strain, and only the first isolate collected was analyzed (subsequent isolates were considered redundant). All isolates with a cefpodoxime MIC of ≥4 μg/ml and a ceftazidime MIC of ≥1 μg/ml were identified as an “ESBL alert” on the MicroScan Walkaway. ESBL confirmatory testing was performed via the Etest method using ceftazidime-ceftazidime-clavulanic acid and cefotaxime-cefotaxime-clavulanic acid strips in accordance with CLSI guidelines (7).Due to the high prevalence of cefoxitin resistance in this population, PCR was performed to detect the presence of a bla_AmpC gene, the product of which can mask the effects of clavulanic acid on the ESBL confirmatory test (18, 27). Since the primers used in this study to identify the bla_AmpC gene (18) have since been shown to amplify the plasmid-mediated bla_CMY gene from Citrobacter freundii (19), it can be inferred that the genes detected were part of the bla_CMY lineage. Salmonella Newport strain 0007-33 was used as the positive control (21). PCR was performed for the genes bla_TEM, bla_SHV, and bla_CTX-M as published previously (9, 10, 27). Salmonella Newport strain 0007-33 was also used as the TEM and SHV positive control (21), and E. coli strain MISC 336 (CTX-M-1 positive) was used as the CTX-M positive control.The bla_TEM, bla_SHV, and bla_CTX-M PCR products were sequenced using both strands of DNA for each PCR product. Protein sequences were aligned using Lasergene software (DNASTAR, Inc., Madison, WI) and included GenBank sequences (http://www.ncbi.nlm.nih.gov/GenBank/index.html) to confirm ESBL genotype. Mutations were evaluated with reference to the Lahey Clinic website (http://www.lahey.org/studies/). GenBank accession numbers used for alignment of protein sequences were {"type":"entrez-protein","attrs":{"text":"AAR25033","term_id":"38606069","term_text":"AAR25033"}}AAR25033 for TEM-1 and {"type":"entrez-protein","attrs":{"text":"ABF29674","term_id":"94502886","term_text":"ABF29674"}}ABF29674 for SHV-2. CTX-M accession numbers were derived from a list on the Lahey Clinic website. Specific primers for the CTX-M-1 group (M13U and M13L) were used to amplify the entire coding sequences of these bla_CTX-M genes (23). Sequencing and analysis were carried out as described above to identify the specific CTX-M subtype.Seventy of the 150 E. coli isolates had an “ESBL alert” on the MicroScan Walkaway, and after removal of redundant isolates, 60 isolates were tested further. ESBL confirmatory testing was positive for six of these 60 isolates (Table ​(Table1,1, column 3).

TABLE 1.

Distribution of β-lactamase and extended-spectrum β-lactamase genes

Absorption,Pharmacokinetics, and Safety of Triclosan after Dermal Administration

We evaluated the pharmacokinetics and safety of the antimicrobial agent triclosan after dermal application of a 2% triclosan-containing cream to six volunteers. Percutaneous absorption calculated from urinary excretion was 5.9% ± 2.1% of the dose (mean ± standard deviation). The amount absorbed suggests that daily application of a standard adult dose would result in a systemic exposure 890 times lower than the relevant no-observed-adverse-effect level. Triclosan can be considered safe for use in hydrophobic creams.Triclosan is an antimicrobial agent with broad-spectrum activity against Gram-positive and Gram-negative bacteria as well as some molds and yeasts. It is bacteriostatic at low concentrations as it blocks lipid synthesis, whereas at higher concentrations (as reached in dermatological preparations) membrane destabilization and triclosan-induced K⁺ leakage lead to a rapid bactericidal effect (9, 16). Furthermore, triclosan potently inhibits the growth of Toxoplasma gondii and Plasmodium (13, 20) and shows anti-inflammatory effects after topical administration (11, 19).For more than 20 years, triclosan has been used widely worldwide in medical and consumer products (5, 21). In dermatological preparations, it is an effective topical antiseptic to reduce colonization with Staphylococcus aureus and to treat superinfected atopic dermatitis (2, 8, 10, 22). Despite the almost-ubiquitous occurrence of the substance, pharmacokinetic studies are sparse.We evaluated the pharmacokinetics and safety of triclosan in a clinical study in six healthy Caucasians. The study was approved by the Ethics Committee of the University of Cologne and by the competent German authorities, and all participants gave their written informed consent. Demographic baseline characteristics are shown in Table ​Table1.1. The study medication (provided by Infectopharm) was a hydrophobic cream containing 2% triclosan. Its composition corresponds to a dermatological standard preparation (NRF 11.122) which is listed in the Neues Rezeptur Formularium (German List of Recommended Standard Formulations). Approximately 60 g of the cream was massaged into the skin of the whole body except for the head and genitals. Exposure was ended by taking a shower 12 h after administration. The subjects were confined to the clinical ward under standardized conditions from 10 h prior until 48 h after study drug administration.

TABLE 1.

Baseline characteristics of all subjects^a

In Vitro Double and Triple Bactericidal Activities of Doripenem,Polymyxin B,and Rifampin against Multidrug-Resistant Acinetobacter baumannii,Pseudomonas aeruginosa,Klebsiella pneumoniae,and Escherichia coli

In vitro double and triple bactericidal activities of doripenem, polymyxin B, and rifampin were assessed against 20 carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance. Bactericidal activity was achieved in 90% of all bacteria assayed using combinations of polymyxin B, doripenem, and rifampin against five each of the carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli isolates studied. Combinations with these antibacterials may provide a strategy for treatment of patients infected with such organisms.Carbapenem resistance in Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli is acknowledged worldwide (1, 2, 4, 13, 15, 16). Mechanisms of carbapenem resistance in these bacteria can be due to a variety of carbapenemases alone and/or β-lactamases with porin protein mutations as well as other contributory strategies (13, 15). The latest carbapenem approved in the United States, doripenem, has demonstrated in vitro activity against a variety of multidrug-resistant (MDR) Gram-negative organisms, which produce well-characterized β-lactamases, and delayed development of resistance to doripenem has been demonstrated (6, 7, 8, 12). Combination therapy with several classes of antibiotics against multidrug-resistant pathogens has revealed increased activity over single agents and delayed development of resistance (14). This investigation studied the in vitro bactericidal activities of double and triple antibiotic combinations using doripenem (D), polymyxin B (PB), and rifampin (R) against Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae because of their progressive resistance to all available agents.Twenty carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance and nonrelated by pulsed-field gel electrophoresis were studied, including five imipenem-resistant K. pneumoniae isolates (two with KPC and three with ACT-1 [AMPC-type] β-lactamases), five A. baumannii isolates (non-MBL or KPC β-lactamases), five P. aeruginosa isolates (one KPC and four non-MBL or KPC β-lactamases), and five E. coli isolates (one KPC-3 and four KPC-2 β-lactamases). Susceptibility of the isolates was initially determined by our clinical microbiology laboratory using the Phoenix system, and the results were confirmed in the infectious disease research laboratory using Etest methodology according to the manufacturer''s specifications (bioMérieux North America). E. coli ATCC 25922 was tested as the control strain.Bactericidal experiments were performed using double and triple antibiotic combinations of polymyxin B plus doripenem, polymyxin B plus rifampin, doripenem plus rifampin, and polymyxin B plus doripenem and rifampin as previously described (17). Time-kill studies were performed at concentrations at 1/4 of their MICs. Doripenem, polymyxin B, and rifampin alone were also tested at 1/4 MIC against each isolate. For bactericidal assays, samples were taken at time zero and 2, 4, 8, and 24 h. Aliquots were serially diluted, and a 10-μl aliquot was transferred onto plates, spread with a loop to minimize carryover to quantify bacterial counts, and incubated at 35°C for 24 h. Bactericidal activity was defined as a ≥3-log CFU/ml decrease in 24 h.Genotypic and phenotypic characteristics for the carbapenem-resistant isolates used in this study are displayed in Table ​Table1.1. All 20 isolates had the following MICs (μg/ml): for rifampin, ranging from 8 to >32; for ertapenem, >32; for doripenem, 1.5 to >32; for imipenem; 6 to >32; for meropenem, 2 to >32; and for polymyxin B, 0.5 to 12. The results of in vitro bactericidal activities and quantitative fold changes with double and triple antibiotic combinations are shown in Table ​Table2.2. Combinations of polymyxin B-doripenem-rifampin at 1/4 MICs for each antibiotic were bactericidal for 4/5 K. pneumoniae, 3/5 A. baumannii, 5/5 P. aeruginosa, and 5/5 E. coli isolates. Combinations of polymyxin B-doripenem at 1/4 MICs for each antibiotic were bactericidal for 1/5 K. pneumoniae, 1/5 A. baumannii, 1/5 P. aeruginosa, and 4/5 E. coli isolates. Combinations of polymyxin B-rifampin at 1/4 MICs for each antibiotic were bactericidal for 1/5 K. pneumoniae, 2/5 A. baumannii, 1/5 P. aeruginosa, and 2/5 E. coli isolates. Combinations of doripenem-rifampin at 1/4 MICs for each antibiotic were bactericidal for 2/5 K. pneumoniae, 2/5 A. baumannii, 1/5 P. aeruginosa, and 1/5 E. coli isolates. Bactericidal activity was achieved in 85% of all bacteria assayed using combinations of polymyxin B-doripenem-rifampin, 30% with polymyxin B-doripenem, 30% with doripenem-rifampin, and 25% with polymyxin B-rifampin at 1/4 MICs. Doripenem, polymyxin B, and rifampin tested alone, at 1/4 MIC, were not bactericidal.

TABLE 1.

Genotypic and phenotypic characteristics for carbapenem-resistant isolates^a

Genetic and Functional Variability of AmpC-Type β-Lactamases from Acinetobacter baumannii

The incidence of naturally occurring AmpC β-lactamases with extended activities toward several cephalosporins was evaluated among 17 ceftazidime (CAZ)-resistant Acinetobacter baumannii isolates. Five AmpC β-lactamases (named ADC β-lactamases) were identified, among which those possessing the Val208Ala (inside the omega-loop) or Asn283Ser (helix H-10) substitution conferred higher levels of resistance (4- to 64-fold higher) to CAZ and to cefotaxime in Escherichia coli. This study demonstrates that peculiar AmpCs playing a role in resistance to broad-spectrum cephalosporins in A. baumannii may be identified.Acinetobacter baumannii is commonly associated with serious nosocomial infections (4, 10, 14). A growing number of β-lactamases conferring resistance to broad-spectrum cephalosporins in Acinetobacter spp. have been identified. Even though resistance to oxyiminocephalosporins (ceftazidime and cefotaxime) may be related to production of extended-spectrum β-lactamases (ESBLs), it is usually associated with overproduction of an AmpC-type β-lactamase (1, 2, 9). Overexpression of the bla_AmpC gene of A. baumannii may occur as a result of an insertion sequence, ISAba1, providing strong promoter sequences (5, 8, 13), being located upstream.Most AmpC-type β-lactamases naturally produced by Gram-negative bacteria hydrolyze amino- and ureidopenicillins, cephamycins (cefoxitin and cefotetan), and, at a low level, oxyiminocephalosporins, such as ceftazidime, cefotaxime, and ceftriaxone, and monobactams, such as aztreonam (3). AmpCs possessing a broad substrate activity have been reported in Enterobacteriaceae and Pseudomonas aeruginosa (11, 12, 16, 19, 20). These AmpCs (or extended-spectrum AmpCs [ESACs]) with activities against several cephalosporins may confer reduced susceptibility to those molecules (6, 11, 12, 19). They differ from “regular” cephalosporinases by amino acid substitutions or insertions/deletions in four specific regions that are all located in the vicinity of the active site: the Ω loop, the H-10 helix, the H-2 helix, and the C-terminal extremity of the protein (15).Recently, we have identified the first ESAC (named ADC-33, according to the numbering of AmpC β-lactamases from A. baumannii [9]) from a single A. baumannii isolate (21). ADC-33, possessing a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω loop), hydrolyzed ceftazidime, cefepime, and aztreonam at high levels (21).The present study aimed to evaluate the extent of the variability of ADC β-lactamases in A. baumannii. Seventeen nonrepetitive A. baumannii clinical isolates that were recovered in 2007-2008 from patients hospitalized at the Bicêtre Hospital and were resistant to ceftazidime were included in this study. Genotyping was performed by using pulsed-field gel electrophoresis (PFGE) according to the instructions of the manufacturer (Bio-Rad, Marnes-la Coquette, France) as previously described (17, 22), and it identified eight distinct clones among the 17 isolates (Table ​(Table11).

TABLE 1.

Features of the A. baumannii isolates studied^d

Susceptibility of Gram-Negative Pathogens Isolated from Patients with Complicated Intra-Abdominal Infections in the United States, 2007-2008: Results of the Study for Monitoring Antimicrobial Resistance Trends (SMART)

During 2007-2008, 1,036 Gram-negative bacilli were isolated from patients with complicated intra-abdominal infections in the United States. Against members of the family Enterobacteriaceae, the most active agents in vitro were ertapenem, imipenem, and amikacin, while the least active agent was ampicillin-sulbactam. Ertapenem and imipenem were active against all extended-spectrum-beta-lactamase (ESBL)-positive Escherichia coli. Antimicrobial resistance in Gram-negative bacilli isolated from patients with complicated intra-abdominal infections in the United States continues to increase.In 2003, the Infectious Diseases Society of America published guidelines on the appropriate selection of antimicrobials for the management of patients with complicated intra-abdominal infections (IAIs) (14). These guidelines addressed the choice of antimicrobials for both empirical and directed antimicrobial therapy for both community-associated and hospital-associated infections/pathogens. Several surveillance studies and reports have documented the increased levels of antimicrobial resistance to many of the recommended agents with significant awareness of the incidence of extended-spectrum-beta-lactamase (ESBL)-positive Escherichia coli and Klebsiella pneumoniae/oxytoca (2, 5, 7, 8, 10). In 2002, the Study for Monitoring Antimicrobial Resistance Trends (SMART) was initiated to monitor yearly the antimicrobial susceptibility of Gram-negative bacilli associated with both community- and hospital-associated IAIs. This report documents the in vitro susceptibility of the most common pathogens associated with IAI in the United States in 2007-2008 to ertapenem, imipenem, and 10 comparator antimicrobial agents.(This work was presented in part as a poster [poster 814] at the 47th Annual Meeting of the Infectious Diseases Society of America, Philadelphia, PA, 29 October to 1 November 2009.)Up to 100 consecutive aerobic or facultative anaerobic Gram-negative bacilli were collected from patients with complicated IAIs from 18 hospital sites in the United States representing all 9 CDC regions in 2007-2008. Antimicrobial susceptibility testing and ESBL detection/confirmation were performed using broth microdilution as previously described (5). Reference strains recommended by the Clinical and Laboratory Standards Institute (CLSI) for quality control were utilized, and MICs were interpreted according to CLSI guidelines (3). A total of 1,036 isolates were collected in 2007-2008 (Table ​(Table1).1). The most commonly isolated pathogens were as follows (percentage of total is shown in parentheses): E. coli (43.1), K. pneumoniae (17.7), Pseudomonas aeruginosa (13.3), Enterobacter cloacae (8.4), Proteus mirabilis (3.0), Klebsiella oxytoca (2.7), Enterobacter aerogenes (2.3), Citrobacter freundii (1.7), and Serratia marcescens (1.5). Overall, members of the family Enterobacteriaceae accounted for 84.7% of all pathogens and 80.4% of the group of pathogens containing only species with greater than 10 isolates represented. Bacteria that were not members of the family Enterobacteriaceae, including P. aeruginosa and Acinetobacter spp., accounted for most of the remaining pathogens.

TABLE 1.

Susceptibility of IAI pathogens in the United States, 2007-2008

In Vitro Activity of Azithromycin against Nontyphoidal Salmonella enterica

The in vitro activity of azithromycin against 1,237 nontyphoidal Salmonella enterica isolates collected from Finnish patients between 2003 and 2008 was investigated. Only 24 (1.9%) of the isolates tested and 15 (5.1%) of the 294 isolates with reduced fluoroquinolone susceptibility had azithromycin MICs of ≥32 μg/ml. These data show that azithromycin has good in vitro activity against nontyphoidal S. enterica, and thus, it may be a good candidate for clinical treatment studies of salmonellosis.Salmonella is one of the most common causes of food-borne illnesses and a major cause of human infections all over the world (23). Salmonella infections are usually treated with fluoroquinolones or extended-spectrum cephalosporins. Unfortunately, excessive use of fluoroquinolones both in human and in veterinary medicine has led to increasing numbers of resistant isolates, including nontyphoidal strains of Salmonella enterica (18, 19, 28). In addition, the nonclassical quinolone resistance phenotype (the Qnr phenotype), showing reduced susceptibility to ciprofloxacin (MIC of ≥0.125 μg/ml) but susceptibility or only low-level resistance to nalidixic acid (MIC of ≤32 μg/ml), has become more common (6, 14, 19, 20, 22). Extended-spectrum β-lactamase (ESBL) producers have emerged in Enterobacteriaceae and in Salmonella, and there are reports of the coappearance of ESBL and qnr genes in the same transferable genetic elements (5, 10, 21, 27). These resistance problems may jeopardize the treatment of severe Salmonella infections. Thus, alternative antibiotics for the treatment of Salmonella infections are needed.Salmonella isolates are intrinsically resistant to erythromycin via active efflux (2) but naturally susceptible to azithromycin (29), which is a 15-membered erythromycin derivative. Resistance to macrolides is usually conferred by mutations in nucleotides A2058 and A2059 of the 23S rRNA, according to the Escherichia coli numbering (26). Also, the alteration of the 50S ribosomal subunit proteins L4 (rlpD) and L22 (rlpV) may lead to macrolide resistance (4).The purpose of the present study was to determine the in vitro activity of azithromycin against nontyphoidal Salmonella isolates collected between 2003 and 2008 from Finnish patients. Special attention was paid to isolates with reduced fluoroquinolone susceptibility or showing the Qnr phenotype. In addition, mutations in the 23S rRNA and in the L4 and L22 ribosomal proteins were investigated.(This work was presented in part at the 19th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Helsinki, Finland, 2009 [P1043].)A total of 1,237 nontyphoidal Salmonella isolates (638 domestic and 599 foreign) collected from Finnish patients between 2003 and 2008 were included in this study. Starting in January each year, we collected the first 100 domestic and the first 100 foreign, i.e., collected from Finnish travelers returning from abroad, Salmonella isolates. The strains were serotyped at the National Salmonella Reference Centre in Finland.The MICs of antimicrobial agents for the isolates were determined by the agar dilution method according to the CLSI guidelines (8). Mueller-Hinton II agar (Becton Dickinson, Cockeysville, MD) was used as the culture medium. All 1,237 Salmonella isolates were tested for susceptibility to ciprofloxacin, nalidixic acid, and azithromycin (all from Sigma, Steinheim, Germany). We tested 809 selected isolates for erythromycin (Sigma) and 635 for telithromycin (Sanofi Aventis, Paris, France) susceptibility. Control strains for susceptibility testing were as described previously (14). On the basis of earlier publications (1, 16, 17), the MIC breakpoint chosen for reduced ciprofloxacin susceptibility was ≥0.125 μg/ml. For nalidixic acid, CLSI breakpoints were used (9). Based on the EUCAST recommendation for S. enterica serovar Typhi (www.eucast.org) and a previous publication (3), the epidemiological cutoff value chosen for azithromycin was ≥32 μg/ml. The susceptibility data were analyzed by using the WHONET 5.4 computer program.Pyrosequencing was used to detect macrolide resistance causing point mutations in the ribosomal target sites A2058 and A2059 (E. coli numbering) of the 23S rRNA in 22 isolates with azithromycin MICs of ≥32 μg/ml, 44 isolates belonging to the Qnr phenotype, and 73 isolates showing erythromycin MICs of ≥32 μg/ml. Pyrosequencing was performed with previously described primers and protocols (15) using a PSQ 96MA pyrosequencer.The 50S ribosomal proteins L4 and L22 were amplified, and mutations were screened in 24 isolates having azithromycin MICs of ≥32 μg/ml, of which 6 were of the Qnr phenotype. In addition, 13 isolates having only erythromycin MICs of ≥32 μg/ml and showing the Qnr phenotype were tested. DNA was prepared as described above. The specific primers used for amplification of the complete L4 and L22 genes rlpD and rlpV were Salm_L4_f (5′-TGAAGGCGTAAGGGGATAGCA-3′) and Salm_L4_r (5′-TCAGCAGA CGTTCTTCACGAA-3′) and Salm_L22_f (5′-GAAATAAGGTAG GAGGAAGAG-3′) and Salm_L22_r (5′-CCATTGCTAGTCTCCAGAGTC-3′). The PCR conditions were as follows: 94°C for 10 min, 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s for 33 cycles. Any L4- or L22-positive results were confirmed by direct sequencing of both strands of amplicons using specific PCR primers as previously described (24). Amino acid sequences were then compared with the known rlpD and rlpV genes by a BLAST search through the European Bioinformatics Institute (http://www.ebi.ac.uk/Tools/blast/).Two different populations of S. enterica were detected regarding the azithromycin MIC distribution. The majority of the isolates had azithromycin MICs of 4 to 8 μg/ml, i.e., representing the wild-type population, whereas a minority of the isolates had MICs of 32 to ≥128 μg/ml, i.e., over the epidemiological cutoff value. Between 2003 and 2008, 24 (1.9%) of the 1,237 isolates had azithromycin MICs of ≥32 μg/ml (Fig. ​(Fig.1;1; Table ​Table1).1). Nine (1.4%) of the 638 domestic isolates and 15 (2.1%) of the 599 foreign isolates had azithromycin MICs over the epidemiological cutoff value.Open in a separate windowFIG. 1.Histograms of azithromycin (n = 1,237), erythromycin (n = 809), and telithromycin (n = 635) MICs for S. enterica isolates collected from Finnish travelers between 2003 and 2008. The vertical line represents the resistance breakpoint.

TABLE 1.

Twenty-four S. enterica isolates showing azithromycin MICs of ≥32 μg/ml

Diversity,Epidemiology, and Genetics of Class D β-Lactamases

Class D β-lactamase-mediated resistance to β-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D β-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of β-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D β-lactamase genes are mostly associated to class 1 integron or to insertion sequences.Class D β-lactamases, also known as oxacillinases or OXA-type β-lactamases (OXAs), are active-serine-site enzymes like Ambler class A and class C β-lactamases, differing from class A and C enzymes in amino acid structure, whereas class B β-lactamases are metalloenzymes with a Zn²⁺ ion(s) in the active site (4, 71, 78). Even though class D includes mostly enzymes with higher hydrolysis rates for cloxacillin and oxacillin than for benzylpenicillin (hence the name oxacillinases), not all class D β-lactamases have this characteristic. Most of the class D enzymes belong to group 2d of the Bush functional classification scheme for β-lactamases (23). Among the four β-lactamase molecular classes, class D β-lactamases are the most diverse enzymes (107). This diversity is observed at both the genetic and biochemical levels, with enzymes possessing either a narrow or expanded spectrum of hydrolysis. In addition, several class D β-lactamases have an expanded spectrum of activity resulting from point mutations.Although many class D β-lactamase genes are embedded into class 1 integrons, recent reports indicated that other specific genetic structures, including insertion sequences and transposons, may be associated with class D β-lactamase genes. Numerous class D β-lactamase genes have been identified as a source of acquired resistance in gram-negative bacteria, but recent studies have shown that class D β-lactamases are also naturally produced in clinically significant pathogens and environmental species (107).This review focuses on the diversity and substrate profiles of class D β-lactamases, their sources, and the genetics of acquisition of the corresponding genes. All the class D β-lactamases for which a sequence is available in the GenBank databases are listed in Table ​Table11.

TABLE 1.

Features of oxacillinases

Tetracycline Susceptibility Testing and Resistance Genes in Isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus Complex from a U.S. Military Hospital

Infections with multidrug-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex bacteria complicate the care of U.S. military personnel and civilians worldwide. One hundred thirty-three isolates from 89 patients at our facility during 2006 and 2007 were tested by disk diffusion, Etest, and broth microdilution for susceptibility to tetracycline, doxycycline, minocycline, and tigecycline. Minocycline was the most active in vitro, with 90% of the isolates tested susceptible. Susceptibilities varied significantly with the testing method. The acquired tetracycline resistance genes tetA, tetB, and tetA(39) were present in the isolates.Multidrug-resistant Acinetobacter baumannii-Acinetobacter calcoaceticus complex bacteria cause infections in traumatic wounds in U.S. military personnel injured in Iraq and Afghanistan (9). A previous study of isolates recovered at our facility during 2003 to 2005 found these isolates to be highly susceptible to minocycline (6), and our clinical experience with minocycline for the treatment of these infections has been favorable (5). The possibility of increasing resistance to minocycline is of concern, as it would further restrict therapeutic options for multidrug-resistant isolates and may necessitate the use of agents with a greater potential for toxicity, such as colistin. Tigecycline has been proposed as a therapeutic option for multidrug-resistant A. baumannii-A. calcoaceticus complex infections (Tygacil package insert, Wyeth Pharmaceuticals, 2005), but its in vitro activity against isolates of military origin is uncertain.The antibiogram for tetracycline, doxycycline, minocycline, and tigecycline was determined by using 133 clinical isolates from blood and wound infections of 89 patients admitted to our facility in 2006 and 2007. Serial isolates were included only when separated by at least 72 h. Susceptibilities to each antimicrobial were determined from a single measurement by disk diffusion (Becton Dickinson, Sparks, MD), Etest (AB Biodisk, Solna, Sweden), and broth microdilution by the methods of the Clinical and Laboratory Standards Institute (CLSI) (3). Discordant results were tabulated as very major errors (reported susceptible when resistant), major errors (reported resistant when susceptible), or minor errors (reported intermediate when resistant or susceptible or vice versa). CLSI interpretive criteria were used (4), except for tigecycline, for which no interpretive criteria are available.Clonal relationships were assessed by pulsed-field gel electrophoresis (PFGE) with ApaI digestion (Centers for Disease Control and Prevention PulseNet protocols; http://www.cdc.gov/PULSENET/protocols.htm [accessed 25 July 2007]), with modifications for Acinetobacter. Strains were considered to be related if their Dice coefficient was ≥85% (Bionumerics; Applied Maths Inc., Austin, TX). The 89 single-patient isolates were confirmed to be members of the A. baumannii-A. calcoaceticus complex by amplification of bla_OXA-51-like β-lactamase genes and by amplified ribosomal DNA restriction analysis (ARDRA) with the restriction enzymes AluI, HinfI, HhaI, RsaI, MboI, and MspI (12, 13).The presence of tetracycline resistance genes in 89 single-patient isolates was determined by PCR amplification for the tetA, tetB, tetH, tetL, tetM, tetA(39), and tetA(41) genes with previously published primer sequences by previously described methods (1, 8, 11). Data were analyzed according to susceptibility phenotype, PFGE type, and Acinetobacter species as determined by ARDRA.Consistent with our clinical experience and prior laboratory observations, minocycline had the highest in vitro activity, reflected in the MICs for 50 and 90% of the strains tested (MIC₅₀ and MIC₉₀, respectively) and the percentage of isolates susceptible according to the broth microdilution method (Table ​(Table1).1). The in vitro activities of tetracycline and doxycycline were low. The modal MIC of minocycline was more favorable than that of tigecycline.

TABLE 1.

Broth microdilution susceptibilities and MIC distribution from 89 single-patient A. baumannii-A. calcoaceticus complex isolates

In Vitro Activities of DC-159a,a Novel Fluoroquinolone,against Mycobacterium Species

The in vitro activities of DC-159a against seven species of Mycobacterium were compared with moxifloxacin, gatifloxacin, levofloxacin, and rifampin. DC-159a was the most active compound against quinolone-resistant multidrug-resistant M. tuberculosis (MIC₉₀, 0.5 μg/ml) as well as drug-susceptible isolates (MIC₉₀, 0.06 μg/ml). The anti-tubercle bacilli activity of DC-159a was 4- to 32-fold more potent than those of currently available quinolones. DC-159a also demonstrated the highest activities against clinically important nontuberculous mycobacteria.To be effective, tuberculosis (TB) treatment with a multidrug regimen must be continued for at least 6 months. This complicated regimen makes TB control difficult because of the long treatment duration, resulting in nonadherence of treatment, the development of multidrug-resistant TB (MDR-TB), and an additional problem of TB and human immunodeficiency virus (HIV) coinfection. Consequently, new anti-TB agents are urgently needed to overcome these problems (14).Quinolones such as ofloxacin and levofloxacin are classified and used as second-line drugs for MDR-TB cases because they inhibit the supercoiling action of DNA gyrase, which is different from the target enzymes of first-line drugs; however, their bactericidal activities against Mycobacterium tuberculosis are weak in clinical use (15). Currently, moxifloxacin and gatifloxacin, which are 8-methoxy fluoroquinolones, have proven to have more potent activities and may enable the duration of treatment to be shortened. Therefore, combination regimens consisting of new quinolones are expected to improve the treatment of TB (3, 11, 13). However, there is a problem of cross-resistance among quinolones caused by the previous usage of old quinolones in the treatment of MDR-TB and other respiratory infections. Thus, the emergence of quinolone-resistant M. tuberculosis is a concern (4).DC-159a is a newly synthesized broad-spectrum 8-methoxy fluoroquinolone. This compound has been shown previously to have potent activities against various respiratory pathogens, including quinolone-resistant strains (2, 7). The present study was performed to compare the in vitro antimycobacterial activities of DC-159a with three currently available quinolones and also rifampin against M. tuberculosis, including quinolone-resistant MDR (QR-MDR) strains and six species of clinically important nontuberculous mycobacteria (NTM).(This work was presented in part at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2006.)Bacterial strains were isolated from Japanese patients between 1997 and 2003. The 32 M. tuberculosis isolates included drug-susceptible isolates (n = 21) and QR-MDR isolates (n = 11; levofloxacin MICs, ≥2 μg/ml; rifampin MICs, ≥6 μg/ml; isoniazid MICs, ≥4 μg/ml). The NTM isolates comprised slowly growing mycobacteria, including M. kansasii (n = 22), M. avium (serovar 4 [n = 10] and serovar 8 [n = 23]), and M. intracellulare serovar 16 (n = 17), and rapidly growing mycobacteria, including M. fortuitum (n = 10), M. abscessus (n = 12), and M. chelonae (n = 10). The bacterial strains were isolated from clinical specimens and cultured in Middlebrook 7H9 broth medium (Difco, MD) supplemented with 10% albumin-dextrose-catalase enrichment and 0.2% glycerol to provide 10⁷-CFU/ml stock cultures and stored at −80°C until use.Antimicrobial agents were obtained as pure substances from their manufacturers: DC-159a and levofloxacin (LVX; both from Daiichi-Sankyo Pharmaceuticals Co., Ltd., Tokyo, Japan), moxifloxacin (MXF; Bayer Yakuhin, Ltd., Osaka, Japan), gatifloxacin (GAT; Kyorin Co., Ltd., Tokyo, Japan), rifampin (RIF; Sigma-Aldrich Co., Tokyo, Japan).MICs were determined according to the standard agar dilution method recommended by NCCLS (10). A multipoint inoculator (Microplanter; Sakuma Seisakusho, Tokyo, Japan) was used to inoculate approximately 10⁴ CFU per 2-μl spot onto Middlebrook 7H10 agar (Difco, MD) supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.5% glycerol. The tested agar plates contained antimicrobial agents ranging from 0.015 to 128 μg/ml in 2-fold dilutions. The inoculated agar plates were incubated at 37°C for 21 days for M. tuberculosis, 14 days for M. avium and M. intracellulare, 7 days for M. kansasii, and 3 days for M. fortuitum and M. abscessus. M. chelonae was incubated at 30°C for 3 days. The MIC was defined as the lowest drug concentration yielding no visible colony formation on the agar surface.Summaries of the in vitro activities of DC-159a against M. tuberculosis and NTM in comparison with those of the other three quinolones and RIF are shown in Tables ​Tables11 and ​and2,2, respectively. MICs of currently available quinolones were generally similar to those of previous reports (1, 5, 8). DC-159a was the most active compound against M. tuberculosis. The MIC₉₀ of DC-159a against drug-susceptible M. tuberculosis was 0.06 μg/ml, which was 2-fold lower than that of RIF and 4- to 8-fold lower than those of other tested quinolones. The MIC₉₀ of DC-159a against QR-MDR M. tuberculosis was 0.5 μg/ml, which was 4- to 32-fold lower than those of other tested quinolones and equal to that of LVX against drug-susceptible M. tuberculosis.

TABLE 1.

Comparison of MICs of DC-159a with other quinolones and rifampin against M. tuberculosis

IncI1 Plasmid Carrying Extended-Spectrum-β-Lactamase Gene blaCTX-M-1 in Salmonella enterica Isolates from Poultry and Humans in France, 2003 to 2008

We report the dissemination of a conjugative IncI1 plasmid carrying bla_CTX-M-1, conferring resistance to extended-spectrum cephalosporins, in Salmonella enterica isolates from poultry and humans in France from 2003 to 2008. By IncI1 plasmid subtyping, this plasmid was shown to be genetically related to that found in Escherichia coli isolates from healthy poultry in France.Food-producing animals are the primary reservoir of zoonotic pathogens, and the prevalence of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and Salmonella enterica has increased in recent years. In Belgium and France, the emergence of resistance to ESCs, due to extended-spectrum β-lactamases (ESBLs), in E. coli and S. enterica from animal (mainly cattle and poultry) and human origins (1, 3, 6, 7, 10, 11, 14, 15, 17) has been reported. Resistance in these bacteria was reported to be conferred mainly by the ESBL genes bla_CTX-M-2, bla_CTX-M-9, bla_CTX-M-15, and bla_TEM-52 (1, 3, 6, 11, 14, 15, 17). In addition, the occurrence of CTX-M-1 ESBL resistance in E. coli isolates recovered from food animals (cattle, poultry, and swine) in France was recently reported (7, 10, 11). The ESBL resistance genes were shown to reside on large conjugative plasmids of the IncI1 or IncHI2 incompatibility group (3, 5, 6).Since 2003, a number of S. enterica strains showing resistance to ESCs by production of an ESBL not previously detected in Salmonella in France and with various additional resistances to other antibiotic families have been isolated from poultry (n = 1) and from humans (n = 9) in France (Table ​(Table1).1). The human isolates consisted of seven serovar Typhimurium (including a monophasic variant) strains, one serovar London strain, and one serovar Newport strain, and the avian isolate was of serovar Llandoff. The purpose of the present study was to identify the ESBL gene and to characterize the plasmid(s) carrying this gene. For this, in addition to other methods, we applied the recently described plasmid multilocus sequence typing method for IncI1 plasmids carrying ESBL genes (5).

TABLE 1.

Characteristics of the Salmonella strains and their transconjugants producing CTX-M-1 used in this study^a

Incidence and Antimicrobial Susceptibility of Escherichia coli and Klebsiella pneumoniae with Extended-Spectrum β-Lactamases in Community- and Hospital-Associated Intra-Abdominal Infections in Europe: Results of the 2008 Study for Monitoring Antimicrobial Resistance Trends (SMART)

From 2002 to 2008, there was a significant increase in extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli isolates in European intra-abdominal infections, from 4.3% in 2002 to 11.8% in 2008 (P < 0.001), but not for ESBL-positive Klebsiella pneumoniae isolates (16.4% to 17.9% [P > 0.05]). Hospital-associated isolates were more common than community-associated isolates, at 14.0% versus 6.5%, respectively, for E. coli (P < 0.001) and 20.9% versus 5.3%, respectively, for K. pneumoniae (P < 0.01). Carbapenems were consistently the most active drugs tested.Gram-negative bacilli (GNB) continue to play an important role as a cause of hospital-associated (HA) infections (8, 19) and have the leading role in intra-abdominal infections (IAIs) (3, 20). Furthermore, reports from the Asia/Pacific region show strikingly high levels of extended-spectrum beta-lactamase (ESBL)-producing isolates of Escherichia coli and Klebsiella pneumoniae and diminished susceptibility to many antimicrobial agents (11, 16). Antimicrobial resistance in European GNB has been reported (4, 7, 10, 17, 25, 27), but none of these studies focused specifically on IAI pathogens. The Study for Monitoring Antimicrobial Resistance Trends (SMART) monitors the susceptibilities of aerobic GNB from IAIs worldwide. This report describes the frequency of occurrence and antimicrobial susceptibilities of ESBL-producing E. coli and K. pneumoniae isolates in community-associated (CA) and HA IAIs in Europe in 2008 and compares these data with those published previously (1, 11, 22).Isolates were derived from intra-abdominal body sites (e.g., appendix, peritoneum, colon, bile, pelvis, and pancreas) and were mostly obtained during surgery, though some paracentesis specimens were also accepted. Isolates were considered to be community associated if they were recovered from a specimen taken less than 48 h after the patient was admitted to the hospital and hospital associated if the specimen was taken 48 or more hours after hospital admission, as described previously (11). Isolates were from 37 hospitals in 12 countries (1 in Estonia, 4 in France, 5 in Germany, 2 in Greece, 4 in Italy, 1 in Latvia, 1 in Lithuania, 3 in Portugal, 9 in Spain, 1 in Switzerland, 3 in Turkey, and 3 in the United Kingdom). Isolates were sent to the central laboratory of International Health Management Associates, Inc. (Schaumburg, IL) for confirmation of identification and antimicrobial susceptibility testing. MICs were determined using MicroScan dehydrated broth microdilution panels (Siemens Medical Solutions Diagnostics, West Sacramento, CA), following Clinical and Laboratory Standards Institute (CLSI) and the panel manufacturer''s guidelines (5). The MIC interpretive criteria followed guidelines established by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org [2009]). Using 2008 CLSI guidelines, E. coli and K. pneumoniae isolates were classified as ESBL producers if there was at least an 8-fold reduction (i.e., three doubling dilutions) of the MIC for ceftazidime or cefotaxime tested in combination with clavulanic acid versus the MIC when tested alone (5). Quality control (QC) testing was performed each day of testing using the CLSI QC strains E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603 (positive ESBL control) (6). Fisher''s exact test (two tailed) was used to determine significance, and the modified Wald method was used to calculate 95% confidence intervals.The percentages of isolates from HA or CA infections were 57.9% and 38.5% for E. coli (P < 0.001) and 70.8% and 23.9% for K. pneumoniae (P < 0.001), respectively. Another 3.6% of E. coli isolates and 5.3% of K. pneumoniae isolates were from patients without information regarding length of stay at the time of specimen collection. After declining slightly in 2004, 2005, and 2006, the rates of ESBL-positive E. coli isolates rose during the last 2 years of the study, reaching 12.3% in 2007 and 14.0% in 2008 (Table ​(Table1).1). The rates of ESBL-positive K. pneumoniae isolates in HA infections were more variable before rising to 20.9% in 2008. ESBL-positive E. coli isolate rates among CA IAIs remained relatively stable, from 4.0% in 2002 to 7.4% in 2007 and 6.5% in 2008. The rate of ESBL-positive K. pneumoniae isolates in CA infections was 5.3% in 2008, lower than the rates observed in 2005, 2006, and 2007 and comparable to that in 2003 (4.4%). However, the generally smaller numbers of ESBL-positive K. pneumoniae isolates led to correspondingly wider 95% confidence intervals. The rates of ESBL-positive E. coli isolates in HA infections ranged from 0% (Lithuania and Switzerland) to 30% (Greece), while those for CA infections ranged from 0% (Latvia and Lithuania) to 24% (Italy) (Table ​(Table2).2). The rate of CA ESBL-positive E. coli isolates in most countries was lower than the HA rate. ESBL-positive E. coli isolates were detected in all countries with the exceptions of Lithuania and Switzerland. The rates of ESBL-positive K. pneumoniae isolates in HA IAIs also ranged widely,with the highest rate of 56.0% observed in Italy, followed closely by 50% in both Latvia and Lithuania. The rates of ESBL-positive K. pneumoniae isolates in CA IAIs were 0% in all cases except for Germany and Spain, where the rates were 18.2% and 5.4%, respectively. It is worthwhile noting that the numbers of E. coli and K. pneumoniae isolates in several countries were small, leading to very broad confidence intervals. Furthermore, the number of investigator sites varied from country to country, with several countries having one site while Spain had nine.

TABLE 1.

Percentages of ESBL-positive E. coli and K. pneumoniae isolates from HA and CA IAIs from 2002 to 2008 in Europe^a

AFN-1252, a FabI Inhibitor,Demonstrates a Staphylococcus-Specific Spectrum of Activity

AFN-1252, a potent inhibitor of enoyl-acyl carrier protein reductase (FabI), inhibited all clinical isolates of Staphylococcus aureus (n = 502) and Staphylococcus epidermidis (n = 51) tested, including methicillin (meticillin)-resistant isolates, at concentrations of ≤0.12 μg/ml. In contrast, AFN-1252 was inactive (MIC₉₀, >4 μg/ml) against clinical isolates of Streptococcus pneumoniae, beta-hemolytic streptococci, Enterococcus spp., Enterobacteriaceae, nonfermentative gram-negative bacilli, and Moraxella catarrhalis. These data support the continued development of AFN-1252 for the treatment of patients with resistant staphylococcal infections.AFN-1252 is an investigational inhibitor of staphylococcal FabI, an essential enzyme that catalyzes the reduction of trans-2-enoyl-acyl carrier protein (trans-2-enoyl-ACP) to acyl-ACP, the final step in each elongation cycle of bacterial fatty acid biosynthesis (1, 7, 10). Enoyl-ACP reductase is known to have four distinct enzyme forms: FabI, FabK, FabL, and FabV (8). FabI is the sole form of enoyl-ACP reductase present in Staphylococcus aureus, Staphylococcus epidermidis, and a few other bacterial species (6, 8, 9). No alternative enzyme or rescue pathway for FabI in staphylococci has been identified, suggesting that FabI is essential to Staphylococcus cell viability and that resistance to FabI inhibitors such as AFN-1252 will not readily emerge with therapy (1).AFN-1252 is being developed by Affinium Pharmaceuticals, Inc. (Toronto, Canada), in both oral and intravenous formulations, for the treatment of antimicrobial-susceptible and -resistant staphylococcal infections, particularly infections caused by S. aureus. The structure of AFN-1252 has been described previously (9). AFN-1252 has demonstrated in vivo efficacy in a murine subcutaneous abscess model using a strain of methicillin (meticillin)-resistant S. aureus (12). The present study was undertaken to assess the in vitro activities of AFN-1252 against recent clinical isolates of staphylococci, as well as other gram-positive cocci and gram-negative bacilli, to demonstrate the full antibacterial spectrum of activity of AFN-1252.Clinically relevant isolates were collected at 12 Canadian hospital laboratories from January to December 2007 as a part of the ongoing CANWARD in vitro surveillance study and shipped to the coordinating laboratory (Health Sciences Centre, Winnipeg, Canada) for identity confirmation and antimicrobial susceptibility testing. Multidrug-resistant staphylococci were defined as those isolates that were resistant to two or more of the agents ciprofloxacin, clindamycin, and gentamicin and included both methicillin-susceptible and methicillin-resistant isolates. Vancomycin-intermediate S. aureus and vancomycin-resistant S. aureus isolates were obtained through the Network on Antimicrobial Resistance in Staphylococcus aureus program (supported under NIAID, NIH, contract no. N01-AI-95359) for testing against AFN-1252.Clinical and Laboratory Standards Institute (CLSI)-specified broth microdilution testing was performed using frozen, in-house-prepared, 96-well panels containing AFN-1252 and comparative agents (3). Dimethyl sulfoxide was used as the solvent and diluent for AFN-1252. AFN-1252 was tested over a doubling-dilution concentration range of 0.008 to 4 μg/ml, and its MICs were recorded following 20 to 24 h of incubation at 35°C in ambient air. MICs were interpreted using CLSI M100-S17 guidelines (2). For the reference strain S. aureus ATCC 29213, AFN-1252 reproducibly demonstrated an MIC of 0.015 μg/ml.AFN-1252 inhibited all isolates of methicillin-susceptible and methicillin-resistant S. aureus and S. epidermidis at concentrations of ≤0.12 μg/ml (Table ​(Table1).1). AFN-1252 demonstrated MIC₉₀s for methicillin-resistant S. aureus and S. epidermidis and multidrug-resistant S. aureus and S. epidermidis (data not shown) of ≤0.008 μg/ml. AFN-1252 was less active in vitro against vancomycin-intermediate S. aureus isolates (n = 12; MIC₉₀, 0.12 μg/ml) and vancomycin-resistant S. aureus isolates (n = 12; MIC₉₀, 0.06 μg/ml) than against vancomycin-susceptible isolates (MIC₉₀, ≤0.008 μg/ml) (data not shown). AFN-1252 was inactive (MIC range, 4 to >4 μg/ml) against nonstaphylococcal gram-positive pathogens and gram-negative pathogens (Table ​(Table11).

TABLE 1.

Activities of AFN-1252 and comparator agents against staphylococci, nonstaphylococcal gram-positive pathogens, and gram-negative pathogens

Pharmacoeconomic assessment of prevention of the postoperative nausea and vomiting syndrome in plastic (esthetic and reconstructive) surgery

A comparative "cost-efficiency" pharmacoeconomic analysis was made for 5 different anti-emitting drugs, i.e. for "Navoban", "Zofran" (intravenous administration, 8 mg; and suppositories, 16 mg) and for "Metipred" (methylprednisolone) used in aesthetic and reconstructive surgeries of 86 patients. All the drugs were found to be effective for the prevention of PONV. Clinically, the efficiency decreased in the following order: "Novoban" and "Zofran" suppositories 16 mg, "Metiored" and "Zofran" intravenous administration 8 mg. As for "cost-efficiency", preference should be given to "Metipred".     

The marriage of conventional cancer treatments and alternative cancer therapies

The terms "alternative" or "unconventional" have been used to describe any therapy used instead of conventional approaches. Conventional approaches, known as "standard" or "traditional" or "biomedical" approaches, have had broad application in Western medicine. Complementary and alternative medicine has been referred to as "integrative," "integrated," or "complementary" when therapies are combined with conventional approaches, such as those for cancer.