Esther Manso

Effects of Caspofungin against Candida guilliermondii and Candida parapsilosis

ABSTRACT The in vitro activity of caspofungin (CAS) was investigated against 28 yeast isolates belonging to Candida albicans (n = 5), Candida guilliermondii (n = 10), and Candida parapsilosis (n = 13). CAS MICs obtained by broth dilution and Etest methods clearly showed a rank order of susceptibility to the echinocandin compound with C. albicans > C. parapsilosis > C. guilliermondii. Similarly, time-kill assays performed on selected isolates showed that CAS was fungistatic against C. albicans and C. parapsilosis, while it did not exert any activity against C. guilliermondii. In a murine model of systemic candidiasis, CAS given at doses as low as 1 mg/kg of body weight/day was effective at reducing the kidney burden of mice infected with either C. albicans or C. guilliermondii isolates. Depending on the isolate tested, mice infected with C. parapsilosis responded to CAS given at 1 and/or 5 mg/kg/day. However, the overall CFU reduction for C. guilliermondii and C. parapsilosis was approximately 100-fold less than that for C. albicans. Our study shows that CAS was active in experimental systemic candidiasis due to C. guilliermondii and C. parapsilosis, but this activity required relatively high drug dosages.

Nosocomial outbreak of systemic candidosis associated with parenteral nutrition.

Eight patients in two surgical units developed systemic candidosis during a 40-day period from June 5 to July 13, 1987 (in five cases Candida albicans was identified). Three of them died. All cases belonged to a group of 27 patients receiving parenteral nutrition (PN), while among the 108 patients who did not receive PN, no cases were observed (p = .000001). Candida was cultured from two PN bags administered to the cases. A specialized nutrition nurse was responsible for the PN compounding and for maintaining administration sets in the two wards involved. An epidemiological investigation, in which 19 uninfected patients who had had PN were used as controls, showed no significant difference between cases and controls except that lipids were more frequently added to bags administered to cases (p = .0005). Furthermore, the bags administered to cases contained a higher average number of multidose constituents (p = .0008) when the comparison was focused on the two days before the onset of symptoms. Given the favorable medium provided by lipids, even a low level contamination of PN solutions during compounding and/or administration could have been responsible for the exposure of cases to multidose vials suggests, although not conclusively, that an extrinsic contamination occurred during compounding. Six isolates of C albicans were available from four cases. C albicans was cultured from the pharyngeal swabs of two physicians and three nurses, including the specialized nutrition nurse.

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays

Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p<0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.

Detection in Italy of two clinical Enterococcus faecium isolates carrying both the oxazolidinone and phenicol resistance gene optrA and a silent multiresistance gene cfr

Sir, In a century in which the issue of emerging antibiotic resistance is being dominated by severe concerns chiefly regarding Gram-negative organisms, the multiresistance gene cfr is probably the greatest emerging problem in Gram-positive pathogens, particularly staphylococci and enterococci. The concern over this problem is motivated not only by the fact that the resistance involves linezolid—widely used in serious infections caused by MDR Gram-positive organisms, often as a last-resort drug—but also, and critically, by the fact that the frequent location of cfr on conjugative plasmids makes the resistance transferable. Now, the report in China of a second plasmid-borne transferable gene, optrA, conferring efflux-mediated oxazolidinone (including second-generation tedizolid) and phenicol resistance in enterococcal isolates adds further to the concern. As soon as the first report, with the sequence of an Enterococcus faecalis plasmid (pE349) carrying optrA (accession no. KP399637), became available as an Advance Access article in the Journal of Antimicrobial Chemotherapy, we decided to test for the optrA gene in 81 Enterococcus isolates from blood samples, which make up the first batch of an enterococcal collection we had recently started for a study including cfr screening. Identification at the species level was performed using VITEK 2 (bioMérieux, Marcy-l’Étoile, France). The cfr and optrA genes were sought by PCR using primer pairs internal to either gene: respectively, the known pair cfr-fw and cfr-rv, yielding a 746 bp amplicon, and the specially designed pair optrA-fw and optrA-rv, which yielded a 422 bp amplicon (Table S1, available as Supplementary data at JAC Online). Two Enterococcus faecium isolates were positive for cfr and two were positive for optrA. Much to our surprise, there were in fact only two positive isolates (E20818 and E35048), since each carried both optrA and cfr. The antibiotic MICs and other features for the two isolates are reported in Table 1. Both isolates had a relatively low linezolid MIC, 4 mg/L, a value that is regarded as ‘susceptible’ according to EUCAST and ‘intermediate’ according to CLSI. Both had a tedizolid MIC of 2 mg/L; breakpoints for resistance have recently been established by EUCAST for staphylococci and b-haemolytic streptococci (.0.5 mg/L) and for viridans group streptococci (.0.25 mg/L). The two isolates were also examined for mutations in 23S ribosomal RNA (not detected) and for the phenicol exporter genes fexA and fexB (not detected). The two isolates exhibited closely related SmaI-PFGE profiles; one (E35048) was investigated for molecular traits. Sequencing demonstrated that optrA and cfr displayed high-level DNA identities (98% and 99%, respectively) to the respective reference sequences (accession numbers KP399637 and AJ57936). Three amino acid changes were detected in the protein sequence of cfr and 21 (4 of which were already reported in Chinese isolates) in the protein sequence of optrA compared with the respective reference sequences. The results of long PCR assays seeking a possible linkage between optrA and cfr were negative. The genetic contexts of both genes proved capable of undergoing excision in circular form, and were completely sequenced. The sequence of the minicircle containing optrA (3350 bp), deposited under accession no. KT892063, included a transposase gene downstream of optrA. This transposase gene exhibited 70% DNA identity and 65% amino acid identity to a chromosomal transposase from Clostridium sticklandii (accession no. FP565809). The minicircle (3405 bp) containing the cfr gene and one intact IS, ISEnfa5, was almost identical to a cfr genetic context described in Staphylococcus lentus (accession no. KF049005). Considering the low MICs of linezolid, florfenicol and chloramphenicol (Table 1), in spite of the prezsence of two resistance genes acting by different mechanisms (cfr perturbing the ribosome function and optrA providing for active efflux), RT–PCR experiments were performed to check the actual transcription of the two genes (Figure S1). We found that optrA was transcribed, whereas cfr was not. Although the exact mechanism of nontranscription is still being investigated, preliminary data indicate a 52 bp deletion in the regulatory region upstream of cfr. Interestingly, a cfr gene failing to mediate resistance to oxazolidinones and phenicols has been described in a porcine E. faecalis isolate in China. Our collection of Enterococcus blood isolates is still in progress, and the overall results of the survey will be assessed and

In vitro activities of ramoplanin and four glycopeptide antibiotics against clinical isolates of Clostridium difficile.

Seventy strains of Clostridium difficile, all isolated from symptomatic patients, were found to be uniformly susceptible to ramoplanin, a new glycolipodepsipeptide antibiotic, and to four glycopeptides (vancomycin, teicoplanin, and two semisynthetic teicoplanin derivatives). Ramoplanin is recommended for further evaluation in the treatment of C. difficile-associated disease.

Heterogeneity of Tn5253-Like Composite Elements in Clinical Streptococcus pneumoniae Isolates

ABSTRACT Several drug resistances in Streptococcus pneumoniae are associated with mobile genetic elements, which are loosely subdivided into a group of smaller (18- to 27-kb) and a group of larger (>50-kb) elements. While the elements of the former group, which typically carry the tetracycline resistance determinant tet(M) and whose prototype is Tn916 (18 kb), have been studied extensively, the larger elements, whose prototype is Tn5253 (∼65.5 kb), are not as well explored. Tn5253 is a composite structure consisting of two independent conjugative transposons, Tn5251 (which is virtually identical to Tn916) and Tn5252 (∼47.5 kb), with the former inserted into the latter. Tn5252, which so far has only partially been sequenced, carries an integrase gene, driving its site-specific insertion into the host cell genome, and the chloramphenicol resistance cat pC194 determinant. This study investigated 20 clinical isolates of S. pneumoniae, which were selected on the basis of cat pC194-mediated chloramphenicol resistance. All 20 isolates harbored a Tn5253-like element. The composite elements (some of which have been completely sequenced) demonstrated considerable heterogeneity that stemmed from a dual variability: in the Tn5252-like element, due primarily to differences in the integrase gene but also to differences in cargo genes and in the overall genetic organization, and in the Tn916-like element, with the possible involvement, besides Tn916, of a number of Tn916 family pneumococcal elements carrying different erythromycin resistance genes. In mating experiments, only one composite element, containing a less typical Tn916 family element, appeared to be nonmobile. Being part of a Tn5253-like composite element may confer on some Tn916-like transposons, which are apparently nontransferable as independent genetic elements, the ability to be mobilized.

Comparative Effects of Micafungin, Caspofungin, and Anidulafungin against a Difficult-To-Treat Fungal Opportunistic Pathogen, Candida glabrata

ABSTRACT The aim of this study was to compare the in vitro and in vivo activities of micafungin, caspofungin, and anidulafungin against Candida glabrata. The MICs against 28 clinical isolates showed that the overall susceptibilities to caspofungin and to micafungin were not statistically different in the absence of human serum, whereas the isolates were less susceptible to micafungin than to caspofungin in its presence. Minimum fungicidal concentrations, as well as time-kill experiments, showed that caspofungin was more active than anidulafungin, while micafungin was superior to either caspofungin or anidulafungin without serum; its addition rendered caspofungin and micafungin equally effective. A murine model of systemic candidiasis against a C. glabrata-susceptible isolate was performed to study the effects of all three echinocandins, and kidney burden counts showed that caspofungin, micafungin, and anidulafungin were active starting from 0.25, 1, and 5 mg/kg of body weight/day, respectively. Two echinocandin-resistant strains of C. glabrata were selected: C. glabrata 30, a laboratory strain harboring the mutation Fks2p-P667T, and C. glabrata 51, a clinical isolate harboring the mutation Fks2p-D666G. Micafungin activity was shown to be as effective as or more effective than that of caspofungin or anidulafungin in terms of MICs. In vivo studies against these resistant strains showed that micafungin was active starting from 1 mg/kg/day, while caspofungin was effective only when administrated at higher doses of 5 or 10 mg/kg/day. Although a trend toward colony reduction was observed with the highest doses of anidulafungin, a significant statistical difference was never reached.

In vitro and in vivo effects of echinocandins against Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis

OBJECTIVES The aim of the present study was to compare, in vitro and in vivo, the effects of caspofungin, micafungin and anidulafungin against Candida parapsilosis complex isolates. METHODS In vitro activities of all three echinocandins were assessed against C. parapsilosis sensu stricto (n = 4), Candida orthopsilosis (n = 4) and Candida metapsilosis (n = 3) using broth microdilution susceptibility testing, minimum fungicidal concentration determination and a killing-curve assay, in the absence and in the presence of 50% human serum. Then, the activities of all drugs were investigated in an immunocompromised murine model of systemic candidiasis. Animals were infected with six isolates (two for each species) and treated with the echinocandins administered at 0.25, 1, 5 and 10 mg/kg/day for six consecutive days. Fungal burdens were assessed in kidney tissues on day 7 post-infection. RESULTS Geometric mean MICs of caspofungin, micafungin and anidulafungin for C. parapsilosis sensu lato were, respectively, 0.09, 0.14 and 0.20 mg/L without serum, and 0.70, 3.92 and 5.84 mg/L with serum. The fungicidal activity of all three echinocandins was variable; however, the addition of serum reduced the fungicidal effects against these species. In vivo studies showed that caspofungin at 5 and 10 mg/kg/day significantly decreased the kidney burdens with respect to the controls for all isolates, while micafungin was active at 5 and/or 10 mg/kg/day only against C. metapsilosis. CONCLUSIONS Our susceptibility testing showed that caspofungin was the most active echinocandin against all three species. Also, caspofungin resulted in significant therapeutic effects for treatments of experimental systemic infections due to the three species, while micafungin was effective only against C. metapsilosis.

Survey of clinical isolates of Staphylococcus aureus for borderline susceptibility to antistaphylococcal penicillins.

On the basis of the MICs of methicillin and oxacillin, 975 clinical isolates ofStaphylococcus aureus were categorized as having resistance, borderline susceptibility or full susceptibility to penicillinase-resistant penicillins (PRPs). The borderline phenotype accounted for 122 isolates (12.5 %), whereas 562 isolates were fully susceptible and 290 resistant; one remaining isolate had resistance to methicillin and borderline susceptibility to oxacillin. Reductions in the MICs of methicillin and oxacillin in the presence of sulbactam were greater in strains with borderline PRP susceptibility than in fully susceptible or resistant isolates. Over 99 % of fully PRP-susceptible strains, 93 % with borderline susceptibility and 71 % of resistant strains were susceptible to ampicillin/sulbactam. The production of β-lactamase, assayed in all strains using nitrocefin as substrate, could be detected without prior induction in 729 strains and after induction only in another 156 strains. With only two exceptions, the β-lactamase negative strains were part of the fully PRP-susceptible group of organisms (88 of 562 isolates). Among the borderline isolates, strong β-lactamase reactions were encountered with particular frequency, but not in all strains and not exclusively in borderline strains. Although associated with the majority of borderline strains, β-lactamase hyperproduction thus did not appear to be an essential feature of the borderline phenotype. The results obtained may have implications for laboratory and clinical medicine, also in the light of recent findings suggesting that other mechanisms besides β-lactamase hyperproduction may account for borderline susceptibility to PRPs.

Efficacy of Caspofungin against Aspergillus terreus

ABSTRACT We investigated the in vitro and in vivo activities of caspofungin against Aspergillus terreus. The drug increased survival and reduced tissue fungal burden in neutropenic mice. Therefore, our data support the role of caspofungin in treating systemic infections due to this emerging pathogen.