Role for Fks1 in the Intrinsic Echinocandin Resistance of Fusarium solani as Evidenced by Hybrid Expression in Saccharomyces cerevisiae

The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.Serious fungal infections have increased in recent years as a consequence of increased immunosuppression associated with human immunodeficiency virus infection, organ and tissue transplants, and aggressive treatments for neoplastic and autoimmune disease. These infections typically are treated with ergosterol biosynthesis-inhibiting azole antifungals such as fluconazole. However, azoles have limitations: their activity is fungistatic, acquired resistance in normally susceptible yeast is not uncommon, and intrinsic low- to high-level resistance is demonstrated by many molds (3, 21). The membrane-disrupting antifungal amphotericin B is generally fungicidal and has broad-spectrum activity, and resistance to it is rare, but its use remains limited due to toxicity. The echinocandins (ECs) caspofungin (CSP), micafungin (MCF), and anidulafungin represent the most recently introduced group of antifungals. Importantly, ECs have fungicidal activity against most Candida species (including azole-resistant strains), fungistatic activity against Aspergillus species, and negligible toxicity (5, 20, 24). ECs act by inhibiting the synthesis of the cell wall polysaccharide β-1,3-glucan (7). This can result in cell lysis or more subtle cell wall changes that enhance susceptibility to innate immunity (25, 41).Acquired EC resistance in susceptible fungi is associated with specific mutations in the integral membrane protein Fks1 (or its paralog Fks2) (7, 35). Fks1 is believed to represent the β-1,3-glucan synthase catalytic subunit, although this has not been formally proven since only crude membrane preparations retain catalytic activity. Most resistance-conferring mutations cluster within so-called hot spot 1, which corresponds, within the model yeast Saccharomyces cerevisiae, to Fks1 residues Phe639 to Pro647 (F639-P647) (1, 2, 6, 7, 13, 18, 19, 26, 33, 34, 40). Fortunately, acquired EC resistance is rare. On the other hand, the intrinsic EC resistance of ascomycetous molds such as Fusarium solani and Scedosporium prolificans, zygomycetous molds such as Rhizopus oryzae, and the basidiomycetous yeasts Cryptococcus neoformans and Trichosporon asahii represents a major limitation to EC clinical use. Many of these fungi have emerged in recent years as important opportunistic pathogens (3, 27, 32, 37), and a contributing factor may be their intrinsic resistance to antifungals, including ECs (10, 22, 30).The basis for intrinsic EC resistance has been investigated in several of these fungi, but it remains unclear (14, 15, 28, 39). Notably, Ha et al. (14) reported that F. solani Fks1, when heterologously expressed in A. fumigatus, conferred a modest but potentially significant fourfold decrease in CSP susceptibility, the basis for which was not explored. However, expression in their system was abnormally low, and A. fumigatus itself exhibits some degree of intrinsic EC resistance (5), which together complicate the interpretation of this result. On the other hand, recent studies examining the basis for the intrinsically low EC susceptibility of Candida parapsilosis strongly implicated its Fks1 sequence; specifically, a hot spot 1 substitution equivalent to P647A (12).FKS1 initially was identified as the S. cerevisiae gene whose null mutation confers susceptibility to the calcineurin inhibitor FK506 (7, 8). This susceptibility results from the requirement for the calcineurin-mediated expression of FKS2; relatedly, single fks1Δ and fks2Δ disruptants are viable, but double disruption is lethal (31). Here, we exploit this FK506 susceptibility to construct Fks1 hybrids, replacing hot spot 1 from S. cerevisiae with that from F. solani (and, for comparison, A. fumigatus and Candida glabrata) to further test the hypothesis that intrinsic EC resistance is mediated by Fks1 sequence.     

Role of pfmdr1 Amplification and Expression in Induction of Resistance to Artemisinin Derivatives in Plasmodium falciparum

Artemisinin and its derivatives are the most rapidly acting and efficacious antimalarial drugs currently available. Although resistance to these drugs has not been documented, there is growing concern about the potential for resistance to develop. In this paper we report the selection of parasite resistance to artelinic acid (AL) and artemisinin (QHS) in vitro and the molecular changes that occurred during the selection. Exposure of three Plasmodium falciparum lines (W2, D6, and TM91C235) to AL resulted in decreases in parasite susceptibilities to AL and QHS, as well as to mefloquine, quinine, halofantrine, and lumefantrine. The changes in parasite susceptibility were accompanied by increases in the copy number, mRNA expression, and protein expression of the pfmdr1 gene in the resistant progenies of W2 and TM91C235 parasites but not in those of D6 parasites. No changes were detected in the coding sequences of the pfmdr1, pfcrt, pfatp6, pftctp, and pfubcth genes or in the expression levels of pfatp6 and pftctp. Our data demonstrate that P. falciparum lines have the capacity to develop resistance to artemisinin derivatives in vitro and that this resistance is achieved by multiple mechanisms, to include amplification and increased expression of pfmdr1, a mechanism that also confers resistance to mefloquine. This observation is of practical importance, because artemisinin drugs are often used in combination with mefloquine for the treatment of malaria.Plasmodium falciparum parasites have developed resistance to conventional antimalarial drugs by various means, including alteration of the enzymes targeted by drugs (8, 23, 24, 32) and mutation or amplification of the genes coding for proteins involved in drug transport (13, 34, 35). One of these proteins, P. falciparum multidrug resistance transporter 1 (PfMDR1), or Pgh1, a P. falciparum homologue of mammalian P-glycoprotein (15, 45), has been implicated in resistance to several structurally different antimalarial compounds. In early studies, exposure of P. falciparum laboratory lines to mefloquine resulted in amplification of the pfmdr1 gene (45), with concomitant increases in resistance to mefloquine (MQ), quinine (QN), and halofantrine (HF) (7, 30, 31, 45). Amplification of pfmdr1 was also observed in field isolates from different geographical locations (4, 34, 35, 42). Increased pfmdr1 copy numbers (CN) in field isolates were associated with higher inhibitory concentrations (IC) of MQ, QN, HF, and artemisinin (QHS) in vitro (34, 46) and were linked to the failure of MQ monotherapy and mefloquine-artesunate combination therapy in studies conducted in Thailand and on the Thai-Cambodian border (2, 35). Furthermore, direct evidence of the role of pfmdr1 in the modulation of parasite susceptibility came from a report where inactivation of 1 of 2 copies of pfmdr1 in the P. falciparum FCB line led to moderate increases in susceptibilities to artemisinin and arylaminoalcohol drugs (39).In addition to gene amplification, several polymorphic positions in the pfmdr1 gene (N86Y, Y184F, S1034C, N1042D, and D1246Y) have been identified in field isolates (14) and have been shown to contribute to altered parasite responses to QN, HF, MQ, chloroquine (CQ), and QHS in vitro (10, 28, 33, 36, 39). In particular, the last three mutations (S1034C, N1042D, and D1246Y) are implicated in increased sensitivity to artemisinin over that of the “wild type” (with S, N, and D) (36). Conversely, significant decreases in susceptibilities to QHS, MQ, and HF are observed when the “wild-type” N at position 1042 is restored (39). These findings are consistent with the early observation that the “wild-type” pfmdr1 allele (with N, Y, S, N, and D) is associated with reduced susceptibilities of the progeny of the genetic cross of the P. falciparum 3D7 and HB3 lines to MQ, HF, QHS, and artemether (10).Although PfMDR1 is clearly implicated in the modulation of parasite responses to antimalarial drugs, including artemisinins, the mechanism of its action is largely unknown. A recent study using heterologous expression of PfMDR1 in Xenopus laevis oocytes demonstrated that some drugs, including HF, QN, and CQ, are substrates for PfMDR1 (38). It is not clear whether artemisinins interact with PfMDR1. Several proteins have been shown to interact with artemisinin. The translationally controlled tumor protein (TCTP) binds to radioactively labeled dihydroartemisinin (5) and is overexpressed in rodent Plasmodium yoelii parasite lines with decreased susceptibility to artemisinin (44). Another protein that may interact with artemisinins is the sarcoplasmic reticulum Ca²⁺ ATPase 6 (PfATP6); this enzyme, when expressed in Xenopus oocytes, was specifically inhibited by artemisinin derivatives containing an endoperoxide bridge (11). In addition, the activity of the enzyme was greatly influenced by the introduction of several mutations (e.g., L263E) (43). Furthermore, analysis of naturally occurring polymorphisms in PfATP6 in field isolates from French Guiana suggested that a polymorphism at codon 769 may be associated with reduced susceptibility of these isolates to artemether in vitro (19). However, subsequent reports failed to detect codon 263 or 769 polymorphisms in the field (12, 27, 48).Although resistance to artemisinins has not been documented in the field, induction of artemisinin resistance in vitro may help in the identification of molecular markers and drug target sites as well as in designing strategies for combating artemisinin resistance when it arises.Several attempts have been made to develop resistance to artemisinin derivatives in P. falciparum (18, 20, 47) in vitro. Inselburg (18) induced resistance to artemisinin by using mutagens, but these lines are no longer available for study. Other attempts to select resistance with increasing drug pressure have led to various endpoints. Jiang (20) produced a 3-fold decrease in susceptibility to sodium artesunate (AS), but resistance proved unstable. Yang et al. (47) achieved 8.9-fold resistance to AS, although few data are available about the stability of the resistance selected or the methods used. A recent study reported the development of stable resistance to QHS and AS in the rodent parasite Plasmodium chabaudi chabaudi (1). Linkage group analysis of the resistant progeny from the same parental parasites identified two nonsynonymous mutations occurring independently in the P. chabaudi putative ubiquitin carboxyl-terminal hydrolase gene (pcubp1, or pcubcth): V739F appeared after selection with artesunate, whereas V770F occurred in progeny selected with CQ (17). No new mutations in PcUBP1 were detected after further selection with QHS. Attempts to develop stable resistance in the P. falciparum NF54 and 7G8 parasite lines were unsuccessful; parasites reverted to the sensitive phenotype after cryopreservation (17).Here we report the selection of resistance to artelinic acid (AL) and to QHS and its derivatives in vitro in several P. falciparum lines of different genetic backgrounds. We also investigated the possible mechanisms involved in the development of resistance. We present evidence that pfmdr1 gene amplification and expression are required in order for some, but not all, parasites to withstand high concentrations of AL or QHS in vitro.     

Biofilm Formation and Effect of Caspofungin on Biofilm Structure of Candida Species Bloodstream Isolates

Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans, C. tropicalis, and C. parapsilosis, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.Candida species are opportunistic pathogens that cause superficial and systemic diseases in critically ill patients (8, 22, 44) and are associated with high mortality rates (35%) and costly treatments (8, 19). They rank among the four most common causes of bloodstream infection in U.S. hospitals, surpassing gram-negative rods in incidence (6, 17).Recent studies suggest that the majority of disease produced by this pathogen is associated with a biofilm growth style (7, 16, 28, 48). Biofilms are self-organized communities of microorganisms that grow on an abiotic or biotic surface, are embedded in a self-produced matrix consisting of an extracellular polymeric substance (14, 15, 55), and when associated with implanted medical devices are commonly refractive to antimicrobial therapy.As opportunistic pathogens, Candida species are able to attach to polymeric surfaces and generate a biofilm structure, protecting the organisms from the host defenses and antifungal drugs (11, 16, 45, 48). Candida biofilms are more resistant than their planktonic counterparts to various antifungal agents, including amphotericin B (AMB), fluconazole, itraconazole, and ketoconazole (20, 38, 50). However, the molecular basis for the antifungal resistance of biofilm-related organisms is not completely understood.The complex architecture of Candida biofilms observed both in vitro and in vivo suggests that morphological differentiation to produce hyphae plays an important role in biofilm formation and maturation (7, 32, 33). Baillie and Douglas demonstrated that although mutant cells fixed in either a hyphal or a yeast form can develop into biofilms, the hyphal structure is the essential element for providing the integrity and multilayered architecture of a biofilm (4). It has been reported that Candida parapsilosis, C. glabrata, and C. tropicalis biofilms are not as large as those generated by C. albicans; however, further structural analysis studies are needed to describe biofilm formation by these organisms (30, 31).The mechanisms responsible for the resistance characteristics displayed by Candida biofilms are unclear. Possible mechanisms include a decreased growth rate; nutrient limitation of cells in the biofilm; expression of resistance genes, particularly those encoding efflux pumps; increased cell density; cell aging; or the presence of “persister” cells in the biofilm (1, 3, 5, 29, 34, 36, 38, 43, 46, 48, 50, 51).The echinocandins are a novel class of semisynthetic amphiphilic lipopeptides that display important antifungal activity. The echinocandins that are presently marketed are caspofungin (CAS), micafungin, and anidulafungin. The echinocandins show considerable efficacy in vitro and in vivo in the treatment of candidemia and invasive candidiasis (25, 27, 42). CAS is the first antifungal agent to be licensed that inhibits the synthesis of β-1,3-glucan, the major structural component of Candida cell walls; glucan synthesis might prove to be a particularly effective target for biofilms (29, 31, 38, 48, 50). The paradoxical attenuation of antifungal activity at high echinocandin concentrations is a phenomenon that usually occurs with C. albicans isolates and appears to be specific to CAS among echinocandins. The cells surviving at high concentrations appear to be subject to some drug effect, showing evidence of slowed growth in the presence of CAS (53, 54). Recent studies have described this effect in Candida species biofilms (24, 37, 47); however, we are not aware of studies that have elucidated the effect of CAS on Candida biofilm structure. The present study was designed to (i) characterize the in vitro biofilm growth of Candida species bloodstream isolates and (ii) use scanning electron microscopy (SEM) to obtain visual evidence of the effect of CAS on biofilm morphology changes associated with paradoxical growth (PG).     

Human Antimicrobial Peptide LL-37 Induces MefE/Mel-Mediated Macrolide Resistance in Streptococcus pneumoniae

Macrolide resistance is a major concern in the treatment of Streptococcus pneumoniae. Inducible macrolide resistance in this pneumococcus is mediated by the efflux pump MefE/Mel. We show here that the human antimicrobial peptide LL-37 induces the mefE promoter and confers resistance to erythromycin and LL-37. Such induction may impact the efficacy of host defenses and of macrolide-based treatment of pneumococcal disease.Macrolides are in widespread use and are recommended as the first line of treatment for community-acquired pneumonia (CAP), except in areas compromised by high rates of macrolide resistance (25). Macrolide resistance is therefore a major concern in the treatment of Streptococcus pneumoniae, a major causative agent of CAP, sinusitis, otitis media, and meningitis. In S. pneumoniae, macrolide resistance is conferred primarily by the methylase ErmB (MIC ≥ 64 μg/ml) or the macrolide efflux pump Mef/Mel (1, 20, 30). Mef/Mel-mediated efflux has been shown to be specific for 14- and 15-membered macrolides (2, 32). Mef is encoded by two variants, either mefE or mefA, with mefE being more prevalent in the United States (2). MefE/Mel is encoded on the mobile genetic element mega (8) and has been shown to be inducible by 14- and 15-membered macrolides (1, 2, 32). MefE/Mel-mediated MICs for erythromycin range from 1 to 32 μg/ml and increase by 4-fold upon macrolide induction, on average (37). The clinical implications of low-level macrolide resistance (1 to 8 μg/ml) are controversial (3, 14, 17, 26). Recent studies provide evidence that infection with low-level macrolide-resistant pneumococci constitutes a risk factor for treatment failure (5, 4, 13, 22, 23). About 35% of all pneumococcal isolates from community-acquired respiratory tract infections in the Unites States are macrolide resistant, and Mef/Mel is present in more than 50% of the resistant isolates (15).Cationic antimicrobial peptides (CAMPs) are small cationic, amphiphilic peptides. The major mammalian CAMPs are cathelicidins and defensins, which are constitutively expressed in macrophages and neutrophils and inducibly produced by epithelial cells and at mucosal surfaces. They play an important role in host defense against bacterial infections and are a major component of the innate immune response (11, 19). Sublethal concentrations of CAMPs are known to alter the levels of bacterial gene expression (9). In this respect, we determined that the sole human cathelicidin, LL-37, could alter mefE and mel expression and that this alters pneumococcal susceptibility to macrolides and LL-37.     

Defining the Role of Mutations in Plasmodium vivax Dihydrofolate Reductase-Thymidylate Synthase Gene Using an Episomal Plasmodium falciparum Transfection System

Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.Of the four species of Plasmodium that commonly cause malaria in humans, Plasmodium vivax is the most widely distributed and can account for up to 80 million cases annually (25). Although P. vivax infections cause less mortality than P. falciparum, they do cause a debilitating disease that contributes to significant morbidity and economic loss in many regions where P. vivax is endemic (25). This is compounded by frequent relapses that can occur many times and for many months after the initial infection (6, 16, 22).Plasmodium vivax parasites are susceptible to most antimalarial drugs. However, over the last 20 years there have been many reports that highlight the significant increase in resistance of P. vivax malaria to chloroquine, the recommended first-line treatment of P. vivax, and/or sulfadoxine-pyrimethamine (SP) (1, 7, 9, 13, 29, 31, 41, 42). Although the determinant(s) for chloroquine resistance in P. vivax remains elusive, a genetic basis for antifolate resistance in P. vivax has been identified as polymorphisms in the P. vivax dihydrofolate reductase (PvDHFR) active site, the same mechanism observed in antifolate resistance in P. falciparum (2, 11, 12, 17, 18, 20, 21, 24, 39).In P. falciparum, point mutations within DHFR have been determined as the cause for antifolate resistance, such as pyrimethamine and cycloguanil resistance (4, 5, 8, 30, 32-36, 44, 45). It has been shown that resistance to antifolates results from the accumulation of mutations in the P. falciparum DHFR, principally A16V, N51I, C59R, S108N or S108T, and I164L.A large number of point mutations have been identified in PvDHFR, and some were reported to be prevalent in many areas of P. vivax endemicity (2, 12, 18, 20, 21, 39). A particular set of mutations (F57L + S58R + T61M + S117T) within the P. vivax DHFR was shown to correlate with SP treatment failures (18, 39) and to confer significant antifolate resistance when transfected into antifolate-sensitive P. falciparum (28). Resistance is due to alterations to the pyrimethamine binding site of PvDHFR that reduces parasite-drug interactions (23, 28). However, the contribution of these PvDHFR mutations to resistance to a variety of antifolates drugs is not clear.Due to the difficulty in maintaining P. vivax in in vitro cultures, most studies of the mutations within PvDHFR have been limited to surrogate biological systems such as yeast and Escherichia coli (17, 19, 24, 38). Although these systems have obvious experimental utility, they are different from Plasmodium in many biological aspects, particularly in membrane structures and transporters that can potentially affect the susceptibility to drugs. A recent publication (28) showed that the pvdhfr-ts quadruple mutant allele (57L + 58R + 61M + 117T) that is episomally expressed in P. falciparum provides significant protection against antifolates. This demonstrated an excellent potential of using P. falciparum as a biological system for the transgenic expression of pvdhfr-ts alleles to assess DHFR-TS interactions with antifolates.We report here the use of this P. falciparum expression system to assess the effect that specific mutations within the P. vivax DHFR have on conventional and new-generation antifolate drugs. The findings improve our understanding of the effect of various mutant pvdhfr alleles observed in the field on the parasite responses to current and new generations of antifolates, improve the prediction of malaria drug treatment outcome, and provide a useful tool for drug development.     

Emergence of Ciprofloxacin-Nonsusceptible Streptococcus pyogenes Isolates from Healthy Children and Pediatric Patients in Portugal

We describe 66 ciprofloxacin-nonsusceptible Streptococcus pyogenes isolates recovered from colonized and infected children. The ParC S79A substitution was frequent and associated with the emm6/sequence type 382 (emm6/ST382) lineage. The ParC D83G substitution was detected in two isolates (emm5/ST99 and emm28/ST52 lineages). One isolate (emm89/ST101) had no quinolone resistance-determining region codon substitutions or other resistance mechanisms. Five of 66 isolates were levofloxacin resistant. Although fluoroquinolones are not used in children, they may be putative disseminators of fluoroquinolone-nonsusceptible strains in the community.Streptococcus pyogenes clinical isolates with reduced susceptibility to fluoroquinolones (1, 3, 8, 14, 16, 18, 27, 29) or with high-level resistance (15, 16, 23-25, 28) have been described previously, and the reduced susceptibility to fluoroquinolones is mediated by point mutations in the quinolone resistance-determining region (QRDR) of the parC gene (3, 16, 18) whereas high-level resistance has been associated with mutations in the QRDRs of both parC and gyrA genes (23, 28). To the best of our knowledge, there are no reports documenting the prevalence and characterization of fluoroquinolone-nonsusceptible S. pyogenes associated with asymptomatic colonization. Since 1999 and 2000, we have been collecting S. pyogenes isolates from pediatric patients from different clinical origins and from carriers for the surveillance of antimicrobial susceptibility and for the epidemiological characterization of the isolates. This study aimed to describe the prevalence of ciprofloxacin-nonsusceptible S. pyogenes isolates from colonized and infected Portuguese children from 1999 to 2006 and to characterize the associated clones and resistance mechanisms.     

Underestimation of Vancomycin and Teicoplanin MICs by Broth Microdilution Leads to Underdetection of Glycopeptide-Intermediate Isolates of Staphylococcus aureus

Broth microdilution was compared with tube macrodilution and a simplified population analysis agar method for evaluating vancomycin and teicoplanin MICs and detecting glycopeptide-intermediate isolates of Staphylococcus aureus. Modal vancomycin and teicoplanin MICs recorded by tube macrodilution and the agar plate assay, which both used inocula of 10⁶ CFU, were significantly higher (2 μg/ml) against a panel of borderline glycopeptide-susceptible and glycopeptide-intermediate methicillin-resistant S. aureus (MRSA) bloodstream isolates compared to broth microdilution (1 μg/ml). Vancomycin and teicoplanin MIC distributions by tube macrodilution and agar testing were also markedly different from those evaluated by broth microdilution. The 20-fold-lower inoculum size used for broth microdilution compared to macrodilution and agar MIC assays explained in part, but not entirely, the systematic trend toward lower vancomycin and teicoplanin MICs by microdilution compared to other methods. Broth microdilution assay led to underdetection of the vancomycin-intermediate S. aureus (VISA) phenotype, yielding only three VISA isolates, for which vancomycin MICs were 4 μg/ml compared to 8 and 19 VISA isolates detected by macrodilution and agar testing, respectively. While macrodilution and agar testing detected 7 and 22 isolates with elevated teicoplanin MICs (8 μg/ml), respectively, broth microdilution failed to detect such isolates. Detection rates of isolates with elevated vancomycin and teicoplanin MICs by macrodilution and agar testing assays were higher at 48 h than at 24 h. In conclusion, the sensitivity of broth microdilution MIC testing is questionable for reliable detection and epidemiological surveys of glycopeptide-intermediate resistance in S. aureus isolates.Since 1997, two major categories of vancomycin resistance in Staphylococcus aureus have been defined. The first category refers to vancomycin-resistant S. aureus (VRSA) clinical isolates with exogenously acquired, vanA-mediated high-level resistance (vancomycin MICs, ≥16 μg/ml) (7, 45); the second category includes vancomycin-intermediate S. aureus (VISA) isolates that developed low-level resistance (vancomycin MICs, ≥4 to <16 μg/ml) via complex, incompletely defined endogenous mechanisms (6, 10, 21, 51). Since VISA isolates are almost uniformly cross-resistant to teicoplanin (21, 30), they are frequently designated glycopeptide-intermediate S. aureus (GISA) (50). In contrast to vancomycin, widely different teicoplanin susceptibility breakpoints have been proposed by different national or international committees, varying from 2 (13) to 8 (10) μg/ml, which leads to a confusing situation.Soon after their initial discovery in Japan (23), it was realized that a large proportion of VISA isolates, referred to as hVISA, show heterogeneous expression of vancomycin-intermediate resistance, including a minority population (perhaps as few as 10⁻⁶ cells) for which the vancomycin MIC is ≥4 μg/ml, while the majority of bacteria are still vancomycin susceptible (vancomycin MICs, ≤2 μg/ml) (10, 21, 22, 24, 51). No mechanistic model explaining heterogeneous expression of glycopeptide resistance has been provided. hVISA/hGISA are assumed to be precursors of VISA/GISA strains, with glycopeptides providing the selective pressure for conversion (2, 14, 22, 24, 33, 39, 44, 55). On the other hand, serial passages on antibiotic-free media frequently lead to gradual dilution and eventual elimination of the resistant subpopulation (2, 21, 24). These data potentially challenge the previously established distinction between hGISA and GISA (21, 29, 51).Despite repeated efforts to create one, there is no standard molecular or phenotypic assay allowing reliable detection of GISA and hGISA clinical or laboratory isolates (5, 30). This situation can be explained by (i) the multifactorial molecular basis of hGISA/GISA phenotypes, which did not reveal any ubiquitous, single, specific molecular marker for their detection (24-27, 41), and (ii) the variable, phenotypic expression of low-level glycopeptide resistance, which is significantly influenced by several technical parameters, including the compositions of liquid or solid test media and varying time frames and inoculum sizes.Standard CLSI-recommended broth microdilution and agar MIC-testing methods (9) were reported to have suboptimal sensitivity for detecting some hGISA isolates (21, 51) because they use relatively small inocula (5 × 10⁴ CFU/well and 1 × 10⁴ CFU/spot, respectively). Accordingly, specifically designed agar screening or population analysis profiles, as well as modified Etest methods, were developed for improved detection of hGISA and GISA by integrating requirements for larger bacterial inocula and longer incubation periods (5, 17, 24, 48, 51, 54, 58, 60). Nevertheless, standardization of these elaborated, labor-intensive susceptibility test methods is difficult (17, 48, 58, 60), and their relationships with standard glycopeptide MIC breakpoints are not well defined. Finally, the recent revisions of vancomycin MIC breakpoints by CLSI (10) and of both teicoplanin and vancomycin MIC breakpoints by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (13), which were based on glycopeptide susceptibility surveys of S. aureus clinical isolates (15, 51, 59), hamper analysis of hGISA/GISA prevalence data reported before 2006.Despite the lack of standardized hGISA detection methods, a number of clinical reports have linked vancomycin therapeutic failure of methicillin-resistant S. aureus (MRSA) infections with the presence of VISA or hVISA isolates or with emergence of vancomycin-intermediate resistance during glycopeptide therapy (3, 8, 24, 25, 33, 36-38, 44, 51, 52). Even higher rates of vancomycin treatment failures were reported for bacteremic patients infected with MRSA isolates for which vancomycin MICs (2 μg/ml) were still in the susceptible range than for those with lower vancomycin MICs (<2 μg/ml) (3, 11, 18-20, 31, 32, 34, 35, 43, 46, 51). An emerging creep of vancomycin and teicoplanin MICs against MRSA in the last decade, which was suggested by large-scale epidemiological studies (19, 28, 47, 51, 56), has been challenged by more recent data (1, 42). Collectively, most of the discrepancies in the clinical and epidemiological results might have resulted from the lack of reliable, sensitive detection methods for hGISA and GISA.During a retrospective surveillance study that explored the prevalence of intermediate glycopeptide resistance in MRSA bloodstream isolates from our institution, we discovered that vancomycin MICs, assayed by the reference macrodilution (tube) method (9), were 2 μg/ml for a vast majority of our nosocomial isolates. Since these MIC estimates were significantly higher than those currently reported in clinical and epidemiological MRSA surveillance studies, in which the modal vancomycin MIC assayed by the broth microdilution (1, 18, 24, 42, 51) or agar dilution (40, 59) method was 1 μg/ml, we evaluated the impacts of three different susceptibility-testing methods, namely, broth microdilution, tube macrodilution, and a simplified population analysis assay, on glycopeptide MIC distributions for our panel of MRSA isolates. A detailed analysis of parameters that potentially contributed to assay-dependent differences in vancomycin and teicoplanin MIC estimates, such as the inoculum size, time of incubation, and medium composition, was performed. A novel approach, combining broth macrodilution and agar testing, is proposed for discriminating glycopeptide-susceptible from hGISA and GISA isolates.     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.     

Evaluation of a DNA Microarray,the Check-Points ESBL/KPC Array,for Rapid Detection of TEM,SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases

Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemase (KPC) are reported increasingly in Gram-negative bacilli (GNB) (5, 6, 17, 18, 25, 30). KPC producers, initially identified in the United States, are now reported worldwide, and illnesses caused by them have become endemic in some regions (25). Isolates expressing KPC enzymes may be reported as susceptible to carbapenems due to heterogeneous and variable levels of expression of β-lactam resistance.The vast majority of ESBLs belong to the TEM, SHV, and CTX-M types (5, 18, 28). These ß-lactamases are encoded by plasmid-located genes and therefore can very easily spread among Enterobacteriaceae (6, 14, 16). More than 160 TEM-type and 110 SHV-type ß-lactamases have been identified worldwide. Amino acid substitutions at many sites in TEM-1 ß-lactamases have been documented, but those at positions 104, 164, 238, and 240 most often lead to an ESBL phenotype (5, 28). As with TEM, SHV-type ESBLs have one or more amino acid substitutions located around the active site compared to SHV-1: substitutions at positions 238 and/or 240 are the most common and are associated with resistance to ceftazidime, cefotaxime, and aztreonam. Less commonly, an alteration at positions 146 or 179 provides ceftazidime resistance (28).Unlike TEM/SHV enzymes, all the CTX-M enzymes are ESBLs (6, 28). More than 80 CTX-M-variants, sharing 71 to 98% amino acid sequence identities, have now been described and are divided now into five groups (groups CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-8, and CTX-M-25) based on amino acid sequence identity (5).Detection of ESBLs is primarily based on phenotypic testing, such as evidencing a synergy image using the double-disk synergy test performed with expanded-spectrum cephalosporins (ESC) and ticarcillin-clavulanic acid disks (3, 10, 23). This test is not always obvious and is usually time-consuming since it requires subculturing or the use of cloxacillin-containing plates to inhibit the naturally occurring and plasmid-mediated cephalosporinases. Unambiguous identification of KPCs by phenotypic methods is relatively difficult (25). Over the last 20 years, alternative strategies aimed at replacing or complementing traditional phenotypic methods have been proposed. Standard PCR and gene sequencing is still the most widely used technique. Other molecular detection techniques for ESBLs and KPC genes have been proposed, but none have been really suited for routine detection (1, 4, 8, 9, 11, 13, 15, 19, 20, 22, 24, 26, 27, 29, 31, 39), since usually only one ESBL/KPC gene is detected at a time. Finally, the presence of narrow-spectrum variants of TEM and SHV types may complicate significantly the molecular detection of TEM/SHV-type ESBLs (28).Microarray technology has recently been developed for the typing of Salmonella isolates (37, 38). This technology has the potential to detect an almost unlimited number of genes within one reaction mixture. Here, a new commercial DNA-based test, the Check-Points ESBL/KPC array, aimed at identifying TEM-, SHV-, and CTX-M-type ESBLs as well as KPC-type carbapenemases, was evaluated by comparing its performance with that of standard PCR on well-characterized reference strains and on 40 ESBL producers isolated at the Bicetre hospital from January to March 2009.