Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus

Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.Currently, vancomycin-resistant Staphylococcus aureus (VRSA) infections are rare. Thus far, nine cases have been documented in the United States (9, 20), and two have been reported in other countries, including Iran (1) and India (22, 27). Despite this rarity, 7 of the 9 U.S. VRSA cases occurred in the metropolitan Detroit, MI, area. All U.S. VRSA isolates demonstrated either unique pulsed-field gel electrophoresis (PFGE) patterns or unique plasmid restriction patterns (20, 33). This suggests that each VRSA isolate, including the 7 from Michigan, acquired resistance independently and was not the result of transmission of a common VRSA strain between patients.VRSA isolates are thought to occur by in vivo transfer of a vanA plasmid from an Enterococcus isolate to an S. aureus isolate. For most of the VRSA cases, a vancomycin-resistant Enterococcus (VRE) isolate was either coisolated from the same body site as the VRSA isolate or was found to colonize the patient at another body site, such as the nares or rectum (Table ​(Table11 shows a summary of VRE isolates from VRSA patients). There is limited evidence that these VRE isolates are the vanA donors. In previous studies (29, 30, 33), we found that certain VRSA isolates carried vanA on a transposon, and in some cases, a plasmid was identified that was identical to the vanA plasmid or transposon in a VRE isolate from the same patient. In VRSA cases 3, 4, and 5, the VRE and VRSA isolates shared the same vanA plasmid. For all other VRSA cases, the VRSA isolates carried the same Tn1546-like element as their associated VRE isolate (10, 33). Hence, it was proposed that the VRSA phenotype occurred by conjugative transfer of a vanA plasmid from VRE to S. aureus (29, 33).

TABLE 1.

Inc18-like vanA plasmid characteristics of VRSA and VRSA-associated VRE isolates from nine VRSA cases in the United States^c

Characteristics and Population Dynamics of Mosaic penA Allele-Containing Neisseria gonorrhoeae Isolates Collected in Sydney,Australia, in 2007-2008

Eighteen hundred Neisseria gonorrhoeae isolates collected in Sydney, Australia, in 2007 and 2008 were examined for mosaic penA alleles that mediated cephalosporin resistance, and the genotypes of the isolates were evaluated. In 2008, there were substantial increases in numbers (from 15 to 85) and proportions (from 1.5 to 10.3%) of mosaic-containing gonococci and major shifts in genotypic patterns, with 10 new genotypes representing 74 of the 85 mosaic-containing isolates and genotypes detected between 2001 and 2005 having disappeared. Enhanced surveillance of gonococcal resistance to cephalosporins is necessary.Neisseria gonorrhoeae isolates collected following failed treatment for gonorrhea with oral extended-spectrum cephalosporins (ESC) possessed a mosaic penA gene that was associated with increased MICs of these antibiotics (1, 30). Penicillin-binding protein 2 (PBP2), encoded by penA, is the major target site for ESC in gonococci, and altered PBP2, when accompanied by polymorphisms in mtrR and porB1b, has a demonstrated role in gonococcal resistance to ESC (4, 7, 31). A small number of subtypes of N. gonorrhoeae with a mosaic PBP2 spread widely in Japan (4), but considerable sequence type (ST) heterogeneity in geographically and temporally diverse populations of mosaic PBP2-containing gonococci (mPBP2GC) was recorded following the dissemination of these subtypes in the Asia-Pacific region (20, 26). Small numbers of mPBP2GC with limited ST distribution were found in San Francisco (12), and “cefixime-resistant” gonococci from Taiwan (27) were of the same ST, 835, as mPBP2GC from Hong Kong responsible for treatment failures with ceftibuten (8).We examined gonococci from the Neisseria Reference Laboratory (NRL) in Sydney, Australia (6), isolated in 2007 and 2008 for the presence of a mosaic PBP2 (24) and compared the findings with our earlier data on mPBP2GC (20, 24, 26). MICs of ceftriaxone, penicillin, and ciprofloxacin (17) for these isolates, N. gonorrhoeae multiantigen STs (NG-MASTs) (10), and auxotypes (5, 6) of the isolates were determined. Control strains (22) included the mPBP2GC strain WHO K and a Hong Kong isolate of ST835 (8), kindly provided by Janice Lo.A sequential sample comprising the last 60 NRL isolates from 2007 and the first 60 isolates from 2008 contained no mPBP2GC from 2007 but six in the 2008 strains that were of NG-MASTs 3158 (n = 4) and 1407 (n = 2) (Table ​(Table1).1). The mPBP2GC were found exclusively among isolates with an extended phenotype defined by chromosomally mediated resistance to penicillin and ciprofloxacin (17), nonsusceptibility to ceftriaxone (defined here by an MIC of 0.016 μg/ml or more), and proline auxotrophy (CMRP/CipR/CefNS/Pro⁻). mPBP2GC from earlier studies (20, 24) and both the control isolates also had this phenotype.

TABLE 1.

Comparison of mosaic PBP2-containing N. gonorrhoeae strains found in Sydney, Australia, in 2007 and 2008 with those detected in 2001 to 2005^a

In Vitro Activities of Eight Antifungal Drugs against 55 Clinical Isolates of Fonsecaea spp.

The in vitro activities of eight antifungal drugs against clinical isolates of Fonsecaea pedrosoi (n = 21), Fonsecaea monophora (n = 25), and Fonsecaea nubica (n = 9) were tested. The resulting MIC₉₀s for all strains (n = 55) were as follows, in increasing order: posaconazole, 0.063 μg/ml; itraconazole, 0.125 μg/ml; isavuconazole, 0.25 μg/ml; voriconazole, 0.5 μg/ml; amphotericin B, 2 μg/ml; caspofungin, 2 μg/ml; anidulafungin, 2 μg/ml; and fluconazole, 32 μg/ml.Fonsecaea spp., anamorph members of the order Chaetothyriales (black yeasts and other melanized fungi), are principal agents of human chromoblastomycosis (16), a chronic cutaneous and subcutaneous infection characterized by slowly expanding skin lesions, a granulomatous immune response, and the presence of meristematic melanized muriform fungal cells in tissue scrapings (4). The last characteristic is a crucial diagnostic indicator that tends to be similar irrespective of the fungal pathogen. Chromoblastomycosis occurs worldwide in tropical and subtropical climates. Fonsecaea spp. are recoverable from environmental sources, so the disease is considered to be of traumatic origin (8, 9). The taxonomy of the genus Fonsecaea has been reviewed recently (12), and on the basis of sequence data, the following three species are recognized: Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica. These species are morphologically identical, but their clinical spectra differ slightly: F. pedrosoi and F. nubica appear to be associated strictly with chromoblastomycosis, whereas F. monophora has also been isolated from brain abscesses, cervical lymph nodes, and bile (4, 13, 18).Therapy for chromoblastomycosis is challenging because there is no consensus regarding the treatment of choice. Several treatment options have been applied, but these tend to result in protracted disease, low cure rates, and frequent relapses (5, 9, 10, 16, 18). The therapeutic outcomes are variable and are allegedly dependent on the site of infection, lesion size, the etiological agent, and the patient''s health status (4). The specific identification of the causative pathogen is important for epidemiological reasons. The vast majority of cases of chromoblastomycosis in which the pathogen has been identified are caused by F. pedrosoi; for example, F. pedrosoi was isolated from 94% (66/69 cases) of patients with chromoblastomycosis in Sri Lanka (2) and from 98% (77/78 cases) of patients with culture-positive chromoblastomycosis in Brazil (17).The present study aimed at determining the in vitro susceptibilities of clinical isolates of Fonsecaea spp. to seven marketed antifungal drugs and the experimental 1,2,4-triazole antimycotic isavuconazole (11).Fifty-five Fonsecaea strains were obtained from the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) and comprised 21 F. pedrosoi strains, 25 F. monophora strains, and 9 F. nubica strains. Fifty isolates originated from patients with chromoblastomycosis, one isolate was recovered from a patient with a cerebral infection, two isolates were from diseased animals, and two isolates were clinical isolates from unknown sources. Seventeen strains came from southern China, 30 from South and Central America, and 8 from other countries (The Netherlands, Spain, Uruguay, Libya, France, United Kingdom). Strain identities were verified by sequencing the ribosomal internal transcribed spacer (ITS), tubulin (TUB1), and actin (ACT1) regions. In vitro susceptibility was determined as described in CLSI document M38-A2 (6). Briefly, the isolates were cultured on potato dextrose agar (35°C) for up to 7 days, and inocula were prepared by gently scraping the surface of the fungal colonies with a sterile cotton swab moistened with sterile physiological saline containing 0.05% Tween 40. Large particles in the cell suspensions were allowed to settle for 3 to 5 min at room temperature, and then the concentration of spores in the supernatant was adjusted spectrophotometrically (530 nm) to a percent transmission in the range 68 to 71, corresponding to 1.5 × 10⁴ to 4 × 10⁴ CFU/ml, as controlled by quantitative colony counts (6). Antifungal drugs were obtained as reagent-grade powders. The final concentrations of amphotericin B (AMB; Bristol-Myers Squibb, Woerden, The Netherlands), itraconazole (ITR; Janssen Research Foundation, Beerse, Belgium), voriconazole (VOR; Pfizer Central Research, Sandwich, United Kingdom), posaconazole (POS; Schering-Plough, Kenilworth, NJ), and caspofungin (CAS; Merck, Sharp & Dohme, Haarlem, The Netherlands) ranged from 0.016 to 16 μg/ml; the fluconazole (FLU; Pfizer) assay range was 0.063 to 64 μg/ml; and the isavuconazole (ISA; Basilea Pharmaceutica International AG, Basel, Switzerland) and anidulafungin (ANI; Pfizer) assay ranges were 0.008 to 8 μg/ml. After 72 h of incubation at 35°C, MICs and minimum effective concentrations (MECs) were determined visually by comparison of the growth in the wells containing the drug with the drug-free control. The MICs of AMB, ITR, VOR, POS, and ISA were defined as the lowest drug concentration that prevented any discernible growth (100% inhibition), whereas for FLU, the MIC was taken as the lowest concentration supporting ≥50% growth inhibition compared to the growth in the control wells. For CAS and ANI, MECs were determined microscopically as the lowest concentration of drug promoting the growth of small, round, compact hyphae relative to the appearance of the filamentous forms seen in the control wells. Quality control strains Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019), and Candida krusei (ATCC 6258) were included in each assay run.The geometric mean MICs, MIC ranges, MIC₅₀s, and MIC₉₀s for the Fonsecaea isolates are presented in Table ​Table1.1. For each drug-species pair, the MIC₅₀ and geometric mean MIC values differed by <1 log₂ dilution step, indicating that in all cases the MIC₅₀ obtained by inspection reasonably reflected the central tendency of the antifungal susceptibility of the population. All isolates had low MICs (MIC₉₀s ≤ 0.5 μg/ml) for POS, ITR, ISA, and VOR; less active drugs (MIC₉₀s ≥ 2 μg/ml) were AMB, CAS, ANI, and FLU. There were no significant differences in the activities of the surveyed drugs against F. pedrosoi, F. monophora, and F. nubica. The MICs obtained in this study were similar to those obtained in other studies of Fonsecaea isolates (1, 3, 7, 14-16, 21).

TABLE 1.

Geometric mean MICs, MIC ranges, MIC₅₀s, and MIC₉₀s obtained by susceptibility testing of antimycotic agents against Fonsecaea isolates

Susceptibility of Urinary Tract Bacteria to Fosfomycin

We evaluated the in vitro activity of fosfomycin against urinary isolates in a region in Greece that exhibits considerable antimicrobial resistance by evaluating retrospectively relevant susceptibility data retrieved from the microbiological library of the University Hospital of Heraklion, Crete, Greece. We examined 578 urinary isolates. In total, 516 (89.2%) were susceptible to fosfomycin; 415 isolates were gram negative, and 101 isolates were gram positive. Fosfomycin appears to exhibit good levels of in vitro activity against the examined urinary isolates.Urinary tract infections (UTIs) are among the commonest types of bacterial infections (13). The antibiotic treatment for UTIs is associated with important medical and economic implications (12, 17).Antibiotic agents such as beta-lactams, trimethoprim, and cotrimoxazole have been used for the treatment of UTIs (1, 22). However, the emergence of resistant uropathogens led to a shift to fluoroquinolones (18, 21, 26, 27), shorter antibiotic regimens (24, 39), and early switch practices (38). Yet, resistance to fluoroquinolones has been reported (14, 23). Additionally, the emergence of uropathogens, mainly Escherichia coli, exhibiting high rates of resistance due to the production of extended-spectrum beta-lactamases (ESBLs) is worrisome (30, 40).Fosfomycin is an old broad-spectrum bactericidal antibiotic agent that inhibits the synthesis of the bacterial cell. Its pharmacokinetic profile encourages its use for UTIs; the mean peak urinary concentration of an oral single dose of 3 g fosfomycin tromethamine occurs within 4 h, while concentrations sufficient to inhibit the majority of the urinary pathogens are maintained for 1 to 2 days (28). Thus, fosfomycin tromethamine has been approved as an oral single-dose treatment for acute uncomplicated cystitis (34, 39). Data from studies evaluating the role of fosfomycin in infections other than UTIs are also encouraging (8-11). We aimed to evaluate the in vitro activity of fosfomycin against urinary isolates isolated from patients in a 700-bed university hospital in Heraklion, Crete, Greece.The data included in our study were retrieved from the database of the microbiological laboratory of the University Hospital of Heraklion, Crete, Greece. We retrospectively tested fosfomycin susceptibility in all clinical urinary isolates that were collected over a 12-month period (January to December 2008). No specific criteria for the selection of isolates to be subjected to fosfomycin susceptibility testing had been set. Only the first isolate of each species per patient could be included in our study.Quantitative urine cultures were performed by standard techniques using Columbia blood and MacConkey agar plates (bioMérieux, Marcy l''Étoile, France). Plates were incubated for 18 to 24 h at 36°C. The bacterial species identification was performed by using standard biochemical methods, the API system, or the Vitek 2 automated system (bioMérieux, Marcy l''Étoile, France) (36). Antimicrobial susceptibility testing was performed using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) (5). Due to the lack of acknowledged fosfomycin breakpoints for bacteria other than Escherichia coli and Enterococcus faecalis (2, 5, 33), we used the fosfomycin breakpoints for E. coli proposed by the CLSI for the remaining evaluated gram-negative isolates, a practice that was also followed by authors of similar studies (6). Similarly, we used the CLSI breakpoint regarding E. faecalis for the other evaluated gram-positive bacteria. Identification of ESBL-producing strains was performed by phenotypic testing based on the synergy between clavulanic acid and extended-spectrum cephalosporins (5). Carbapenemase production was detected by the modified Hodge test (29).A total of 578 clinical urinary isolates were included. Two-hundred seven (35.8%) of these 578 isolates originated from adult outpatients, 167 (28.8%) from patients hospitalized in medical wards, 74 (12.8%) from adult patients hospitalized in surgical wards, 17 (2.9%) from intensive care unit adult patients, 9 (1.5%) from adult patients in renal replacement therapy clinics, and 14 (2.4%) from patients from areas other than the above-mentioned hospital units. Ninety (15.5%) of the 578 isolates originated from pediatric patients. Eighty-six (95.5%) of these 90 isolates originated from pediatric inpatients.The 578 tested urinary bacterial isolates represented 456 (78.8%) gram-negative isolates and 122 (21.1%) gram-positive isolates. The 456 gram-negative isolates represented 404 (88.5%) members of the Enterobacteriaceae, 266 (65.8%) of which were Escherichia coli, and 52 (11.5%) of which were gram-negative, nonfermentative bacilli. The 122 tested gram-positive isolates consisted of 74 (60.6%) Enterococcus faecalis isolates, 16 (13.1%) Staphylococcus saprophyticus isolates, 13 (10.6%) Staphylococcus aureus isolates, 12 (9.8%) Enterococcus faecium isolates, 6 (4.9%) Streptococcus agalactiae isolates, and a single (0.8%) Staphylococcus epidermidis isolate. In total, 516 (89.2%) of the 578 tested urinary isolates were found to be susceptible to fosfomycin; 415 were gram negative, and 101 were gram positive. Specific data are presented in Table ​Table1.1. The above-mentioned group of 516 fosfomycin-susceptible urinary isolates included specific categories of resistant isolates. Specific data are presented in Table ​Table22.

TABLE 1.

Susceptibility to fosfomycin of gram-negative and gram-positive urinary isolates tested

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

Carbapenem-Resistant KPC-2-Producing Escherichia coli in a Tel Aviv Medical Center, 2005 to 2008

All of the carbapenem-resistant Escherichia coli (CREC) isolates identified in our hospital from 2005 to 2008 (n = 10) were studied. CREC isolates were multidrug resistant, all carried bla_KPC-2, and six of them were also extended-spectrum beta-lactamase producers. Pulsed-field gel electrophoresis indicated six genetic clones; within the same clone, similar transferable bla_KPC-2-containing plasmids were found whereas plasmids differed between clones. Tn4401 elements were identified in all of these plasmids.Carbapenem resistance in Escherichia coli is usually attributed to the acquisition of β-lactamases such as AmpC (14, 23, 24, 27, 31), metallo β-lactamases (4, 17, 25, 33), or KPC-type carbapenemases (2, 3, 26, 32). In 2005, KPC-2-mediated carbapenem-resistant E. coli (CREC) clinical strains were first identified in our hospital (21).The increasing prevalence of carbapenem-resistant Enterobacteriaceae in Israel (30), along with concerns regarding the emergence of highly epidemic clones, led to the study of carbapenem resistance in E. coli in our hospital. We determined CREC prevalence, elucidated the molecular mechanisms contributing to carbapenem resistance, and explored the molecular epidemiology and plasmids associated with this resistance.All of the CREC isolates identified in our hospital from February 2005 to October 2008 were included in this study. Strains were identified as resistant to at least one carbapenem using the Vitek-2 and agar dilution (MIC of imipenem or meropenem, >4 μg/ml; MIC of ertapenem, >2 μg/ml). Antibiotic susceptibilities were determined by Vitek-2 (bioMérieux Inc., Marcy l''Etoile, France), and MICs of carbapenems were determined by agar dilution according to the Clinical and Laboratory Standards Institute (CLSI) protocols (8). MICs of tigecycline and colistin and MICs of imipenem, meropenem, and ertapenem lower than 0.5 μg/ml were determined by Etest (AB Biodisk, Solna, Sweden). The criteria used for the interpretation of carbapenem MICs were based on the CLSI 2010 guidelines (9). The interpretive criterion used for tigecycline was based on FDA breakpoint values for Enterobacteriaceae that define a MIC of ≤2 as susceptible.β-Lactamases were analyzed by analytical isoelectric focusing (IEF) (16) on crude enzyme preparations from sonicated cell cultures as described elsewhere (21). The following β-lactamases were used as controls: TEM-1, pI = 5.4; TEM-26, pI = 5.6; K1, pI = 6.5; SHV-1, pI = 7.6; P99, pI = 7.8; ACT-1, pI = 9.The genetic relatedness between isolates was determined using pulsed-field gel electrophoresis (PFGE) as previously described (7). DNA macrorestriction patterns were compared according to the Dice similarity index (1.5% tolerance interval) (9a) using GelCompar II version 2.5 (Applied Maths, Kortrijk, Belgium). A PFGE clone was defined as a group of strains showing >85% banding pattern similarity (19).Multilocus sequence typing (MLST) was performed on two representative E. coli clones according to the protocol at the E. coli MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).Epidemiological links and potential contact between patients were analyzed using data on room location, consulting physicians, and other procedures performed during their hospitalization.PCR molecular screening of β-lactamase genes and Tn4401 elements (18) was performed using the primers listed in Table ​Table1.1. PCR products were sized on an agarose gel and sequenced using an ABI PRISM 3100 genetic analyzer (PE Biosystems). Nucleotide and deduced protein sequences were identified using the BLAST algorithm (www.ncbi.nlm.nih.gov/).

TABLE 1.

Primers used in this study

Comparison of Anidulafungin MICs Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Method (M27-A3 Document) and Etest for Candida Species Isolates

Anidulafungin Etest and CLSI MICs were compared for 143 Candida sp. isolates to assess essential (within 2 log₂ dilutions) and categorical agreements (according to three susceptibility breakpoints). Based on agreement percentages, our data indicated that Etest is not suitable to test anidulafungin against Candida parapsilosis and C. guilliermondii (54.4 to 82.4% essential and categorical agreements) but is more suitable for C. albicans, C. glabrata, C. krusei, and C. tropicalis (87.9 to 100% categorical agreement).The echinocandins are available for intravenous treatment of Candida infections, especially for patients with recent azole exposure (17, 26, 27). The Clinical and Laboratory Standards Institute (CLSI) has established guidelines and an interpretive susceptibility breakpoint (≤2 μg/ml) for testing echinocandins against Candida spp. (11, 12). We evaluated the suitability (essential and categorical agreements) of anidulafungin Etest MICs for 143 Candida sp. bloodstream isolates from the Hospital La Fe, Valencia, Spain. Categorical agreement was evaluated according to CLSI (11), Garcia-Effron et al. (21), and Desnos-Ollivier et al. (13, 14) susceptibility microdilution breakpoints (≤2, ≤0.5, and ≤0.25 μg/ml, respectively).The 143 isolates included caspofungin-resistant (six heterozygous and homozygous C. albicans mutants, one C. krusei isolate), caspofungin-susceptible (Table ​(Table1),1), and quality control (QC; C. parapsilosis ATCC 22019 and C. krusei ATCC 6258) isolates (8, 15, 23); anidulafungin MICs were within the established QC limits (12).

TABLE 1.

Echinocandin MICs for eight reference isolates of C. albicans and one of C. krusei determined by the CLSI M27-A3 broth microdilution and Etest methods^a

Comparative In Vitro Susceptibilities of Human Mycoplasmas and Ureaplasmas to a New Investigational Ketolide,CEM-101

MICs were determined for an investigational ketolide, CEM-101, and azithromycin, telithromycin, doxycycline, levofloxacin, clindamycin, and linezolid against 36 Mycoplasma pneumoniae, 5 Mycoplasma genitalium, 13 Mycoplasma hominis, 15 Mycoplasma fermentans, and 20 Ureaplasma isolates. All isolates, including two macrolide-resistant M. pneumoniae isolates, were inhibited by CEM-101 at ≤0.5 μg/ml, making CEM-101 the most potent compound tested.Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma fermentans, and Ureaplasma spp. isolates are responsible for infections in the respiratory and urogenital tracts (17, 18). Macrolides have historically been the treatments of choice for M. pneumoniae respiratory infections of adults and children because they have the advantages of being safe and well tolerated in oral formulations and of possessing antiinflammatory properties independent of their antibacterial activity and activity against other microorganisms that may cause clinically similar illness. These properties have also made macrolides attractive for empirical treatment, since mycoplasmal infections are rarely confirmed by microbiological testing. Macrolides are also active against some other Mycoplasma spp., as well as Ureaplasma spp. M. fermentans and M. hominis, however, are resistant to some members of this class, such as erythromycin, but are susceptible to clindamycin (19).During the past several years, concerns have arisen over the impact of the widespread use of macrolides on antimicrobial resistance in respiratory pathogens, such as Streptococcus pneumoniae isolates, among which 30% or more of clinical isolates are no longer susceptible to macrolides and may not respond to treatment with these drugs (7). Recent publications from Japan have confirmed the emergence in 10 to 33% of M. pneumoniae isolates of macrolide resistance that may have implications for patient outcomes (8, 10, 11, 14). These isolates typically have mutations in domain V of the 23S rRNA gene and erythromycin MICs of 32 to >64 μg/ml. A recent report from Shanghai, China, documented that 39 of 50 (78%) M. pneumoniae strains isolated there were macrolide resistant (6). Macrolide-resistant M. pneumoniae has also been reported from France (12) and the United States (20, 21). The Centers for Disease Control and Prevention recently described 3 of 11 cases (27%) of M. pneumoniae infections from an outbreak in Rhode Island that were macrolide resistant (20). We have encountered two children in Birmingham, AL, with macrolide-resistant M. pneumoniae infections of the lower respiratory tract who did not respond initially to treatment with azithromycin and required several days of hospitalization (21). Fluoroquinolone resistance has been described in genital mycoplasmas (1, 3), and tetracycline resistance may now exceed 40% in some populations (17). Azithromycin resistance associated with clinical treatment failure has also been documented in M. genitalium (4). These findings clearly indicate the need for new drug classes or improvements in drugs of existing classes for treatment of mycoplasmal and ureaplasmal infections.CEM-101 is a new ketolide with activity against many bacteria that cause respiratory and/or urogenital infections, such macrolide-resistant S. pneumoniae, chlamydiae, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae (2, 5, 9, 13). To investigate further the antimicrobial spectrum of CEM-101, we studied its vitro activities against human mycoplasmas and ureaplasmas in comparison to the activities of other antimicrobial agents (Table ​(Table11).

TABLE 1.

In vitro activities of CEM-101 and other antimicrobials against Mycoplasma and Ureaplasma species isolated from humans

Sitafloxacin Activity against Helicobacter pylori Isolates,Including Those with gyrA Mutations

Sitafloxacin showed MICs of less than or equal to 0.5 μg/ml against 105 isolates of Helicobacter pylori, including 44 isolates with mutations in the gyrA gene. The highest MICs for garenoxacin and levofloxacin were 8 and 64 times, respectively, higher than the highest MICs observed for sitafloxacin.The guidelines for the management and treatment of Helicobacter pylori infections established by the European Helicobacter Study Group Third Masstricht Consensus Report recommend an eradication antimicrobial chemotherapy consisting of amoxicillin, clarithromycin, and a proton pump inhibitor alone or in combination with metronidazole and clarithromycin (10). On the other hand, a trend toward increased clarithromycin resistance in Japan has been reported (8); furthermore, high metronidazole resistance rates associated with H. pylori eradication failure have been seen in the United States, Europe, and Asia with the exception of Japan (11). In the search for alternative eradication treatment regimens, it has been recently reported in the United States and Europe that levofloxacin may be efficacious in H. pylori eradication therapy (7, 13). At the same time, the increased use of levofloxacin-based eradication regimens has led to increasing resistance to levofloxacin in H. pylori as a result of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene correlating with the decreased effectiveness of levofloxacin in eradication regimens (16). Sitafloxacin is a recently developed fluoroquinolone with wide-spectrum activity, ranging from gram-positive cocci to gram-negative bacilli (1, 15). We studied the effect of mutations in the gyrA gene and its impact on the antimicrobial activity of sitafloxacin in H. pylori.(This study was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)A total of 105 H. pylori isolates were recovered from the gastric mucosa of patients presenting with gastroduodenal diseases in health care facilities in Japan between 2004 and 2005. Of the 105 patients, 57 (54.3%) were males and 48 (45.7%) were females, and the average age was 57.9 years (range in age from 21 to 84). None of the patients had previously undergone eradication therapy. The spectrum of peptic ulcers included 39 (37.1%) cases of chronic gastritis, 21 (20.0%) gastric ulcers, 18 (17.1%) duodenal ulcers, 9 (8.6%) gastric cancers, 8 (7.6%) gastroduodenal ulcers, and 10 (9.5%) cases with other causes or an unspecified diagnosis. Only one isolate per patient was included among the 105 isolates.Susceptibilities to sitafloxacin (Daiichi Sankyo, Japan), garenoxacin [a novel des-fluoro(6)quinolone (Astellas, Japan) (6)], and levofloxacin (Daiichi Sankyo, Japan) were determined by agar dilution method according to CLSI guidelines by using drugs with known potency (4, 5). The agar dilution method was performed by serial twofold dilution on Mueller-Hinton agar (Becton Dickinson, MD) with 5% sheep blood using 1 to 3 μl of a McFarland 2.0-adjusted inoculum and incubation at 35 ± 2°C for 72 h under microaerophilic conditions. For quality control, H. pylori ATCC 43504 was tested with each run.PCR amplification and sequence analysis of the QRDR of gyrA was performed as previously described by Nishizawa et al. (12). The specific primers used for amplification were GYRA-F (5′-TTTRGCTTATTCMATGAGCGT-3′) and GYRA-R (5′-GCAGACGGCTTGGTARAATA-3′). For sequencing, the ABI Prism 3130xl genetic analyzer (Perkin-Elmer, ABI, CA) was used. Sequences were compared to that of the H. pylori wild-type strain (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"L29481","term_id":"508470","term_text":"L29481"}}L29481).Forty-four of the 105 H. pylori isolates exhibited mutations in the gyrA gene (Table ​(Table1).1). Mutations at Asn87 were observed in 14 isolates, while mutations were seen at Asp91 in 25 isolates. The remaining five isolates had mutations in other regions. Table ​Table22 shows the effect of changes in the gyrA gene in the QRDR and its effect on the MICs of sitafloxacin, garenoxacin, and levofloxacin. Mutations involving Asn87 resulted in a shift to higher MIC levels of the drugs than mutations in other regions. Sitafloxacin demonstrated the narrowest MIC distribution with a MIC of ≤0.5 μg/ml against all isolates. In contrast, the highest MICs for garenoxacin and levofloxacin were 8 and 64 times, respectively, higher than the highest MIC observed for sitafloxacin. A scattergram depicting sitafloxacin MICs versus levofloxacin or garenoxacin MICs for 105 isolates with or without gyrA mutations is shown in Fig. ​Fig.1.1. From the scattergram, the increase in garenoxacin and levofloxacin MICs relative to the sitafloxacin MICs is observed. With respect to levofloxacin-resistant isolates with MICs ranging from 2 to 32 μg/ml, garenoxacin and sitafloxacin demonstrated MICs ranging from 0.125 to 4 μg/ml and ≥0.015 to 0.5 μg/ml, respectively.Open in a separate windowFIG. 1.Scattergram of sitafloxacin MICs versus levofloxacin or garenoxacin MICs for 105 Helicobacter pylori isolates. The red and black numbers indicate the frequency of isolates with and without gyrA mutations, respectively.

TABLE 1.

Genetic characteristics of Helicobacter pylori isolates in this study

In Vitro Activity of Thimerosal against Ocular Pathogenic Fungi

The in vitro activity of thimerosal versus those of amphotericin B and natamycin was assessed against 244 ocular fungal isolates. The activity of thimerosal against Fusarium spp., Aspergillus spp., and Alternaria alternata was 256 times, 512 times, and 128 times, respectively, greater than that of natamycin and 64 times, 32 times, and 32 times, respectively, greater than that of amphotericin B. Thimerosal''s antifungal activity was significantly superior to those of amphotericin B and natamycin against ocular pathogenic fungi in vitro.The problem of keratomycosis in developing countries like China is more acute because of its higher incidence and the unavailability of effective antifungals (18, 28, 30). To date, only fluconazole and natamycin are commercially available for ocular use in China. Fluconazole has high bioavailability against Candida spp., but Fusarium spp. and Aspergillus spp. are resistant to it (3, 27, 29). Fusarium spp. and Aspergillus spp. are more commonly associated with keratomycosis, while Candida spp. are rarely implicated as etiological agents of keratomycosis in China (23, 26). Natamycin is the only topical ophthalmic antifungal compound approved in the United States (14). Natamycin is poorly soluble in water. After topical application, natamycin penetrates the cornea and conjunctiva poorly and effective drug levels are not achieved in either the cornea or the aqueous humor (15); it is therefore useful only in the treatment of superficial keratomycosis. Due to the relative unavailability of effective antifungals, keratomycosis fails to resolve in many of the patients who receive antifungal treatment; some patients experience vision loss and eventually corneal perforation, ultimately require penetrating keratoplasty, or even enucleation or evisceration (20, 28). Therefore, it is very important and urgent to explore broad-spectrum antifungals to effectively suppress a wide variety of ocular fungal pathogens and to develop new antifungal eye drops to combat this vision-threatening infection.Thimerosal is a preservative commonly used in ophthalmic solutions, otic drops, topical medicine, and vaccines because of its bactericidal property. However, the efficacy of thimerosal against ocular pathogenic fungi has not been evaluated so far. The present study was performed to determine the antifungal activity of thimerosal versus those of amphotericin B and natamycin against ocular pathogenic fungi in vitro. To our knowledge, this is the first study to determine the antifungal activity of thimerosal against main ocular pathogenic fungi. Results obtained in this study may contribute to the development of new antifungal eye drops.Two hundred forty-four strains of fungi isolated from patients with keratomycosis from the Henan Eye Institute in Zhengzhou, China, were investigated. These isolates were identified based on morphology by standard methods (22, 23, 25). They included 136 Fusarium isolates, 98 Aspergillus isolates, and 10 Alternaria alternata isolates. Candida parapsilosis ATCC 22019 was used as quality control for each test.Thimerosal (Yili Pharmaceutical Co. Ltd., Beijing, China), amphotericin B (Bristol-Myers Squibb, Princeton, NJ), and natamycin (Yinxiang Biotechnology Co. Ltd., Zhejing, China) were studied. They were all dissolved in 100% dimethyl sulfoxide. The stock solutions were prepared at concentrations of 400 μg/ml for thimerosal and 1,600 μg/ml for amphotericin B and natamycin. Drug dilutions were made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid. Final concentrations ranged from 0.0078 to 4 μg/ml for thimerosal and from 0.0313 to 16 μg/ml for amphotericin B and natamycin.A broth microdilution method was performed by following the Clinical and Laboratory Standards Institute M38-A2 document (13). The final inoculum was 0.4 × 10⁴ to 5 × 10⁴ CFU/ml. Following incubation at 35°C for 48 h, the MIC was determined as the lowest concentration of amphotericin B, natamycin, or thimerosal that prevented any discernible growth.The MIC range and mode, the MIC for 50% of the strains tested (MIC₅₀), and the MIC₉₀ were provided for the isolates with the SPSS statistical package (version 13.0). For calculation, any high off-scale MIC was converted to the next higher concentration.The in vitro activities of thimerosal, amphotericin B, and natamycin against the isolates are summarized in Tables ​Tables11 and ​and2.2. When comparing the MIC₉₀s of thimerosal with those of natamycin and amphotericin B, the activity of thimerosal against Fusarium spp. is 256 times greater than that of natamycin and 64 times greater than that of amphotericin B, the activity of thimerosal against Aspergillus spp. is 512 times greater than that of natamycin and 32 times greater than that of amphotericin B, and the activity of thimerosal against Alternaria alternata is 128 times greater than that of natamycin and 32 times greater than that of amphotericin B. Therefore, thimerosal''s effect was significantly superior to those of amphotericin B and natamycin against main ocular pathogenic fungi in vitro.

TABLE 1.

In vitro susceptibilities of ocular Fusarium isolates to thimerosal, amphotericin B, and natamycin

In Vitro Activity of Micafungin against Planktonic and Sessile Candida albicans Isolates

Planktonic and sessile susceptibilities to micafungin were determined for 30 clinical isolates of Candida albicans obtained from blood or other sterile sites. Planktonic and sessile MIC₉₀s for micafungin were 0.125 and 1.0 μg/ml, respectively.Candida albicans device-related infections are associated with growth of organisms in a biofilm state (3, 6). Device removal is often considered necessary for cure (10), since antimicrobial agents have been considered to have poor activity against microbial biofilms. If, however, antimicrobial agents were active against microbial biofilms, device removal might be avoidable.Cell walls are integral to C. albicans biofilms; therefore, antifungal agents that target cell wall synthesis may be active against fungal biofilms (1). We previously showed that caspofungin and anidulafungin had MIC₉₀s of 2 and ≤0.03 μg/ml, respectively, against 30 C. albicans isolates in biofilms (7, 12). We also demonstrated that caspofungin was active in vivo in an experimental intravascular catheter infection model (13).Herein, we evaluated the activity of micafungin against planktonic and sessile forms of the 30 clinical isolates of C. albicans against which we had previously studied caspofungin, anidulafungin, amphotericin B deoxycholate, and voriconazole (7, 12). One isolate per patient was included; isolates were included only if ≤3 types of organisms were cultured from the specimen from which C. albicans was isolated. Isolates were from blood cultures (n = 10), peritoneal fluid (n = 6), abscess fluid (n = 5), soft tissue (n = 5), bone (n = 2), pleural fluid (n = 1), and urine (n = 1). C. albicans GDH 2346 was used as a positive control.Planktonic MICs were determined using broth microdilution (5). Isolates were grown on Sabouraud dextrose agar for 24 h at 37°C. C. albicans was titrated to 76.6% transmittance at 530 nm in sterile saline and then diluted 1/1,000 in RPMI. Serial twofold micafungin dilutions ranging from 16 to 0.03 μg/ml were assayed. Drug dilution and titrated organism (100 μl each) were placed into corresponding wells of a 96-well, round-bottomed microtiter plate and incubated at 37°C. Forty-eight hours later, MICs were read using a reading mirror and scored according to CLSI guidelines. The lowest concentration associated with a ≥50% reduction in turbidity compared with that for the positive-control well was reported as the MIC. Planktonic MICs for micafungin ranged from ≤0.03 to 0.25 μg/ml (Table ​(Table1).1). The MIC₅₀ and MIC₉₀ were 0.125 μg/ml. The GDH 2346 MIC was 0.06 μg/ml.

TABLE 1.

Comparison of planktonic and sessile susceptibilities of 30 C. albicans isolates^a

BEL-2, an Extended-Spectrum β-Lactamase with Increased Activity toward Expanded-Spectrum Cephalosporins in Pseudomonas aeruginosa

A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ß-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The bla_BEL-2 gene was identified from a P. aeruginosa isolate clonally related to another bla_BEL-1-positive isolate.Extended-spectrum ß-lactamases (ESBLs), such as TEM, SHV, PER, VEB, GES, and more recently, CTX-M variants, are reported increasingly to be found in Pseudomonas aeruginosa in various areas (1, 2, 7, 8, 10-12, 15, 17, 21, 23, 27, 28, 30). The BEL-1 ß-lactamase, distantly related to other ESBLs, was identified from a P. aeruginosa isolate from Roeselare, Belgium, which interestingly shows resistance to ticarcillin and ceftazidime but only reduced susceptibility to piperacillin, cefepime, cefpirome, and aztreonam (24). The bla_BEL-1 determinant was found as a gene cassette in the chromosome-borne class 1 integron, In120, that includes other resistance genes (aacA4, aadA5, and smr2) and that was part of a Tn1404-type transposon structure (24). Very recently, Bogaerts et al. (5) reported on the diffusion of BEL-1-producing isolates in various hospital centers of Belgium and also found that BEL-1 could be associated with other relevant β-lactamases, such as the VIM-1 metallo-β-lactamase (5).P. aeruginosa isolate 531 (this study) was recovered from a urine sample of a patient hospitalized in Roeselare, Belgium, in February 2007 for pneumonia and was resistant to all β-lactams but imipenem (Table ​(Table1).1). A synergy between aztreonam or ceftazidime and clavulanic acid-containing disks suggested the synthesis of an ESBL (19). PCR followed by sequencing using ESBL gene-specific primers (24) identified a novel gene encoding BEL-2, which differs from BEL-1 by a single amino acid substitution (Leu to Phe at Ambler position 162) (3). Transfer of a ß-lactam resistance marker from P. aeruginosa 531 to Escherichia coli or to P. aeruginosa reference strains was unsuccessful by either conjugation or transformation (25). Plasmid extraction performed as described previously (14) did not identify any plasmid, suggesting a chromosomal location of the bla_BEL-2-like gene in P. aeruginosa 531. A pulsed-field gel electrophoresis (PFGE) analysis (4) showed that isolates 531 (BEL-2 positive) and 51170 (BEL-1 positive), recovered from the same geographical area, were clonally related. A PCR mapping approach confirmed the presence of a class 1 integron whose structure was identical to that of In120 of P. aeruginosa 51170 (24) and identified an identical structure in P. aeruginosa 531 (data not shown). Overall, these data suggest that the bla_BEL-2 sequence likely resulted from a mutational event that had occurred in In120-carrying P. aeruginosa strains.

TABLE 1.

MICs of β-lactams^a

Emergence of Quinolone-Resistant Bordetella pertussis in Japan

Six Bordetella pertussis strains isolated from children in Japan from 2004 to 2006 showed high-level resistance to nalidixic acid (NAL; MIC, >256 μg/ml) and decreased susceptibilities to fluoroquinolones. All of the NAL-resistant strains had the same D87G mutation in gyrA.Pertussis is an acute respiratory tract infection caused by Bordetella pertussis that is particularly serious for neonates and infants (2, 5, 19). Although the introduction of whole-cell and acellular vaccines caused a drastic decrease in the incidence of pertussis globally compared with that in the prevaccine era, developed countries have experienced a marked increase over the past 15 years (2). There has also been a recent shift in the age distribution of pertussis patients to adults and adolescents, an unrecognized but significant source of infection for neonates and infants (2, 5, 9, 19). Macrolides are widely used for antimicrobial treatment and postexposure prophylaxis of pertussis (2, 5). However, several erythromycin-resistant strains of B. pertussis have emerged in the United States (13, 14, 20) and alternative therapeutic agents are being sought to combat these strains. Several fluoroquinolones demonstrate excellent in vitro activity against B. pertussis (1, 3, 8, 11, 16, 22), and although contraindicated for children (1, 16), they might be candidate agents to treat adults with pertussis.However, as we found six nalidixic acid (NAL)-resistant B. pertussis strains isolated from 2004 to 2006 in Japan by disk diffusion test (no inhibitory zone detected around a 30-μg NAL disk on Bordet-Gengou agar), the MICs of erythromycin, NAL, norfloxacin, ciprofloxacin, sparfloxacin, levofloxacin, gatifloxacin, and chloramphenicol were determined by Etest (AB Biodisk, Solna, Sweden) on Bordet-Gengou agar (7, 10). The quality control strains used were B. pertussis CCUG 30837^T (= ATCC 9797^T) and Staphylococcus aureus ATCC 29213 (10). Strains for which the MICs of erythromycin and NAL were less than or equal to 0.12 and 32 μg/ml were considered susceptible (4, 10). Whole sequences of gyrA, gyrB, parC, and parE of the NAL-resistant strains were determined by PCR and direct sequencing. To prepare template DNA, one loopful colony of each strain was resuspended in 100 μl of 20 mM Tris-2 mM EDTA buffer (pH 7.5), incubated at 100°C for 15 min, and then centrifuged for 5 min at 23,200 × g. The 50-μl reaction mixture contained 2 μl of the supernatant fluid, 25 pmol of each primer, a 0.4 mM concentration of each deoxynucleoside triphosphate, 2.5 U of TaKaRa LA-Taq polymerase (TaKaRa, Shiga, Japan), and LA-Taq GC buffer I (TaKaRa). After an initial denaturation at 95°C for 5 min, amplification proceeded for 30 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 2 min 30 s, with a final 10-min extension at 72°C. Following cleanup with a QIAquick PCR purification column (Qiagen, Valencia, CA), the PCR product was sequenced by using a Big Dye terminator (version 3.1; Applied Biosystems, Foster City, CA). The primers used for PCR and direct sequencing are listed in Table ​Table11.

TABLE 1.

Primers used in this study

Does the Activity of the Combination of Imipenem and Colistin In Vitro Exceed the Problem of Resistance in Metallo-β-Lactamase-Producing Klebsiella pneumoniae Isolates?

Using time-kill methodology, we investigated the interactions of an imipenem-colistin combination against 42 genetically distinct Klebsiella pneumoniae clinical isolates carrying a bla_VIM-1-type gene. Irrespective of the imipenem MIC, the combination was synergistic (50%) or indifferent (50%) against colistin-susceptible strains, while it was antagonistic (55.6%) and rarely synergistic (11%) against non-colistin-susceptible strains (with synergy being observed only against strains with colistin MICs of 3 to 4 μg/ml). The combination showed improved bactericidal activity against isolates susceptible either to both agents or to colistin.During the past decade, VIM metallo-β-lactamases (MBLs) have spread rapidly among Enterobacteriaceae (4). MBL producers commonly exhibit a multiple-drug resistance phenotype as a result of combined chromosomally encoded or plasmid-mediated resistance mechanisms. Frequently, colistin and tigecycline remain the only therapeutic choices. Tigecycline has demonstrated in vitro activity against MBL producers (18), but evidence of in vivo efficacy against a variety of clinical infections (i.e., bacteremia or pneumonia) is still limited. On the other hand, randomized controlled trials supporting the use of colistin as a single-drug regimen, as well as studies on its pharmacokinetic/pharmacodynamic properties, are lacking (11). Recently, the emergence of colistin resistance among Klebsiella pneumoniae isolates further jeopardized the already limited treatment options in the intensive care unit setting (2). For all these reasons, combination therapies are frequently used in clinical practice, especially in hospitals with high rates of infections by MBL producers.(Some of these data were presented at the 45th Infectious Disease Society Annual Meeting, 2007 [15a].)We investigated the in vitro activities of imipenem and colistin alone and in combination against 42 unique clinical isolates of MBL-producing K. pneumoniae isolated in Greek hospitals from February 2004 to September 2006. MICs were determined by Etest (AB Biodisc, Solna, Sweden) and interpreted according to CLSI breakpoints for imipenem (3) and EUCAST breakpoints for colistin (7). The presence of a bla_VIM gene was confirmed by PCR (15). On the basis of PCR-restriction fragment length polymorphism analysis (9), all isolates carried a bla_VIM-1-type gene. Extended-spectrum β-lactamase production was detected with a modified CLSI confirmatory test (8). Genetic relatedness among studied isolates was evaluated with repetitive extragenic palindromic PCR methods (10). Patterns that differed by more than one amplification band were characterized as different. In vitro interactions between imipenem and colistin were tested using time-kill methodology. Antibiotic concentrations used were 10 μg/ml for imipenem (Merck, Rahway, NJ) and 5 μg/ml for colistin sulfate (Sigma, St. Louis, MO) because these concentrations represent the steady state achievable in human serum during treatment (12, 16) and thus are clinically relevant. For susceptible strains, if 4× MIC was not higher than 10 or 5 μg/ml for imipenem or colistin, respectively, this concentration was also tested.Synergy was defined as a ≥2-log₁₀ decrease in CFU/ml between the combination and the most active single agent at the different time points, with the number of surviving organisms in the presence of the combination being ≥2 log₁₀ CFU/ml below the number of organisms in the starting inoculum. Antagonism was defined as a ≥2-log₁₀ increase in CFU/ml between the combination and the most active single agent. All other interactions were characterized as indifferent. Bactericidal activity of single antibiotics or combinations was defined as a ≥3-log₁₀ reduction in the CFU/ml of the initial inoculum after 24 h of incubation (1, 6). The lower limit of detection was 1.6 log₁₀ CFU/ml. For analysis of the results, isolates were classified into four groups according to susceptibility to imipenem and colistin. The chi-square test was used to compare proportions of killing activity or synergy between groups by using Yates continuity correction in two-by-two tables. P values of <0.05 were considered statistically significant.The results are shown in Table ​Table1.1. The imipenem-colistin combination exhibited synergy against 12 of 24 (50%) colistin-susceptible MBL-producing K. pneumoniae isolates tested, but it was antagonistic against 10 of 18 (55.6%) non-colistin-susceptible isolates. Of note, isolates showing colistin MICs of 3 to 4 μg/ml behaved more like colistin-susceptible isolates, since in two of them (50%) a synergistic interaction was noted after exposure to the combination.

TABLE 1.

MICs (μg/ml) of imipenem and colistin against bla_VIM-1-type MBL-producing K. pneumoniae isolates and in vitro interaction of the combination

First Survey of Metallo-β-Lactamases in Clinical Isolates of Pseudomonas aeruginosa in a German University Hospital

A total of 489 clinical isolates of Pseudomonas aeruginosa was investigated for metallo-β-lactamase (MBL) production. Molecular analysis detected a bla_VIM-1 gene in the chromosome of one isolate and a bla_VIM-2 gene carried on the plasmid in seven isolates. Moreover, we showed that an initial screening by combined susceptibility testing of imipenem and ceftazidime followed by a confirmatory EDTA combination disk test represents a valid alternative to the molecular investigation of MBL genes, making MBL detection possible in routine diagnostic laboratories.Metallo-β-lactamase (MBL)-producing Gram-negative bacteria are an increasing public health problem worldwide because of their resistance to all β-lactams except aztreonam (3). MBL genes are typically part of an integron and are either carried on transferable plasmids or are part of the bacterial chromosome (28). The most common transferable MBL families include the VIM-, IMP-, GIM-, SPM-, and SIM-type enzymes which have been detected primarily in Pseudomonas aeruginosa but were also found in other Gram-negative bacteria, including nonfermenters and members of the family Enterobacteriaceae (22). Recently, two new subgroups of MBLs, designated NDM-1 and DIM-1, were identified in a clinical isolate of Klebsiella pneumoniae in India and in a clinical isolate of Pseudomonas stutzeri in the Netherlands, respectively (21, 31).In most studies, reduced susceptibility to imipenem has been adopted as the sole criterion for further phenotypic or molecular investigations in order to detect MBLs (11, 19, 20, 23). However, this criterion seems to be suboptimal, as it does not allow exclusion of isolates characterized by the loss of the OprD porin, the most common mechanism of resistance to imipenem in P. aeruginosa (14, 22). Moreover, several phenotypic tests for detecting MBLs, such as the MBL Etest (20, 25), EDTA combination disk test (20, 25, 30), EDTA disk synergy test (10), and imipenem lysate MBL assay (27), have been developed and evaluated. However, the performance of each of these tests seems to be strongly affected by the local rate of MBL-producing isolates.In Germany, VIM-, and GIM-type enzymes in P. aeruginosa isolates have already been detected (1, 8, 26). Recently, Elias et al. described a nosocomial outbreak caused by a bla_VIM-2-positive P. aeruginosa in patients of the Department of Urology of the university hospital of the University of Würzburg, Würzburg, Germany, between November and December 2007 (4). However, to the best of our knowledge, no systematic surveys of the occurrence of MBLs in clinical isolates of P. aeruginosa have been conducted in Germany so far.Accordingly, this study was designed with the following aims: (i) to develop improved screening criteria for the detection of MBL-producing P. aeruginosa; (ii) to determine the proportion of MBL-producing isolates in clinical isolates of P. aeruginosa in the university hospital of the University of Würzburg in Germany; (iii) to assess the relatedness of MBL-producing isolates; (iv) to determine the locations of the MBL genes detected; and (v) to evaluate two phenotypic tests as confirmatory tests for the detection of MBL production.Since no standard imipenem MIC breakpoints for MBL producers are available, we first analyzed the MICs of imipenem in 10 well-characterized P. aeruginosa control strains shown previously to produce IMP-, VIM-, GIM-, SIM-, and SPM-type enzymes (Table ​(Table1).1). Identification to the species level was confirmed using Vitek 2 GN cards (bioMérieux, Nürtingen, Germany). Antimicrobial susceptibility testing was carried out with Vitek 2 AST-N021 and AST-N110 cards (bioMérieux). MICs were interpreted as recommended by the CLSI (2). All MBL-producing positive-control strains were intermediate sensitive or resistant to imipenem (MIC ≥ 8 μg/ml). Subsequently, we investigated 26 consecutive nonreplicate clinical isolates of P. aeruginosa with an imipenem MIC of ≥8 μg/ml for the presence of MBL genes by multiplex PCR as described by Ellington et al. (5). All clinical isolates were collected in our laboratory throughout 2007 before the outbreak at the university hospital of the University of Würzburg, Würzburg, Germany (4), and all isolates were shown by multiplex PCR to be negative for MBL genes. Since the loss of the OprD porin, the most common mechanism of resistance to imipenem in P. aeruginosa, does not confer ceftazidime resistance (15), we analyzed the ceftazidime MICs in the MBL-producing positive-control strains and in the 26 MBL-negative isolates. While all 10 MBL-producing positive-control strains were resistant to ceftazidime (MIC ≥ 32 μg/ml), only 6 of the 26 MBL-negative isolates were resistant to ceftazidime (P < 0.01) (Table ​(Table1).1). Consequently, we adopted MIC breakpoints for the initial screening of MBL producers of ≥8 μg/ml for imipenem and ≥32 μg/ml for ceftazidime.

TABLE 1.

Comparison of imipenem and ceftazidime MICs in well-characterized MBL-producing positive-control strains and in MBL-negative clinical isolates^a

Detection of KPC in Acinetobacter spp. in Puerto Rico

During an island-wide PCR-based surveillance study of beta-lactam resistance in multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus-baumannii complex isolates obtained from 17 different hospitals, 10 KPC-positive Acinetobacter isolates were identified. DNA sequencing of the bla_KPC gene identified KPC-2, -3, and -4 and a novel variant, KPC-10. This is the first report of a KPC-type beta-lactamase identified in Acinetobacter species.The Acinetobacter calcoaceticus-baumannii complex has been recognized, during the last few decades, as an important opportunistic pathogen associated with life-threatening nosocomial infections and hospital outbreaks, as well as an agent of serious infections in injured U.S. military personnel returning from the Middle East war zones (9, 25, 33). In addition to their ability to survive in adverse environmental conditions (14), the organisms have intrinsic and acquired mechanisms of antimicrobial resistance, such as porin downregulation, overexpression of efflux pumps, chromosomal and plasmid-acquired beta-lactamases, aminoglycoside-modifying enzymes, and fluoroquinolones resistance, among others (11). Class B metallo-beta-lactamases and class D oxacillinase with carbapenemase activity have been identified in Acinetobacter species as a mechanism of broad-spectrum beta-lactam resistance (11, 20).The molecular class A beta-lactamases of the KPC family are a group of potent carbapenemases identified initially in a Klebsiella pneumoniae isolate from the United States and later in other members of the Enterobacteriaceae family and in other geographical regions worldwide (17, 20). Pseudomonas aeruginosa positive for the bla_KPC gene has been recently identified in Colombia, Puerto Rico, and Trinidad and Tobago (3, 28, 29).Up to date, eight different KPC variants (KPC-2 to -9) have being identified differing by 1 or 2 two amino acid substitutions. KPC-2 and -3 are the most common variants identified in Enterobacteriaceae and P. aeruginosa. KPC-6, -7, and -8 have been identified only in K. pneumoniae, while KPC-9 was detected in Escherichia coli and KPC-5 in P. aeruginosa. All the KPC variants except for KPC-7 and -9 have been detected in Puerto Rico (21-24, 29). In this report, we describe for the first time the presence of the KPC gene in clinical isolates of Acinetobacter species in Puerto Rico and the identification of a novel KPC variant, KPC-10, in one of the isolates.A PCR-based surveillance study of beta-lactam resistance was started in January 2009 in 17 hospitals across the island. After Institutional Review Board approval, the participating hospitals sent isolates of all unique, consecutive, multidrug-resistant Acinetobacter species with their corresponding susceptibility report and basic epidemiologic information. PCR screening with family-specific beta-lactamase primers for KPC and IMP and multiplex PCR for OXA carbapenemases and for CTX-Ms were performed as previously described (29, 31, 32). The Vitek DCS-R5 system confirmed the identification of the isolates as those in the A. calcoaceticus-baumannii complex. Bidirectional sequencing of the full-length bla_KPC gene PCR product was independently generated at least twice to identify the type of the KPC variant. DNA sequencing was commercially performed by Davis Sequencing. Sequence alignment and analysis were done online by utilizing the BLAST program (www.ncbi.nlm.nih.gov). Trek GNXF Gram-negative MIC plate microdilution panels (Westlake, OH) were utilized to perform antibiotic susceptibility tests by following the manufacturer''s instruction, and the results were interpreted as recommended by the Clinical and Laboratory Standards Institute (8). No attempts were made to evaluate patients'' therapies or clinical outcomes.From a total of 274 multidrug-resistant Acinetobacter isolates collected from January to May of 2009, 10 (3.4%) were identified as KPC positive. Seven were detected in the metropolitan San Juan area, two in the north, and one in the central region of Puerto Rico.Table ​Table11 shows the patients'' pertinent clinical information and type of KPC variants identified. There were six male and four female patients, whose average age was 69 years, ranging from 40 to 95 years. Six were isolated from patients in the intensive care units either from sputum (four isolates) or blood (two isolates) samples. The average hospital length of stay was 41.6 days (range, 11 to 96 days). Six patients had ventilator-associated pneumonia with or without sepsis. Six patients had skin and soft-tissue infections. All patients had significant comorbid conditions, such as cardiovascular, renal, neurologic, or traumatic injuries. The crude mortality rate for the 10 patients was 40%. Nine patients were treated with multiple antibiotic courses during their hospitalization (data not shown). Our patients shared similar characteristics to those described in the literature, such as significant comorbidities, prolonged hospitalizations, invasive procedures, exposure to multiple antibiotics regimes, and high crude mortality rates (1, 2, 10, 27). In our island-wide surveillance study, all hospitals with KPC-positive Acinetobacter isolates also had KPC-positive Klebsiella pneumoniae, Pseudomonas aeruginosa, and/or Escherichia coli isolates (21).

TABLE 1.

Pertinent clinical information and type of KPC variants