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.     

Role of AbeS,a Novel Efflux Pump of the SMR Family of Transporters,in Resistance to Antimicrobial Agents in Acinetobacter baumannii

In this study, a chromosomally encoded putative drug efflux pump of the SMR family, named AbeS, from a multidrug-resistant strain of Acinetobacter baumannii was characterized to elucidate its role in antimicrobial resistance. Expression of the cloned abeS gene in hypersensitive Escherichia coli host KAM32 resulted in decreased susceptibility to various classes of antimicrobial agents, detergents, and dyes. Deletion of the abeS gene in A. baumannii confirmed its role in conferring resistance to these compounds.Acinetobacter baumannii is an important nosocomial pathogen frequently reported to be associated with a variety of infections, including respiratory tract infections, urinary tract infections, bacteremia, and skin and skin structure infections (3). Reports of the increased isolation of multidrug-resistant A. baumannii clinical isolates from different regions of the United States are appearing at a startling rate (1, 4, 10, 25, 30, 33).Antibiotic resistance in A. baumannii has been attributed to either intrinsic or acquired mechanisms (21). The resistance mechanisms in A. baumannii are diverse and include enzymatic modification of antibiotics, target gene mutation, altered outer membrane permeability, and upregulated multidrug efflux pump activity (20).Efflux systems involve transport proteins that function to reduce the concentration of drugs or toxic substances by transporting them across the inner and outer membranes into the external medium (24). These multidrug efflux systems are classified into five different families: ATP-binding cassette (ABC), major facilitator super family (MFS), resistance/nodulation/cell division (RND), multidrug and toxic-compound extrusion (MATE), and the small multidrug resistance (SMR) family of bacterial integral membrane proteins (22). ABC transporters are ATP-driven efflux pumps; MFS, RND, and SMR are proton driven; and MATE transporters consist of an Na⁺/H⁺ drug antiporter system (22, 23). Genome sequence analyses reveal that, on average, efflux pumps constitute at least 10% of the transporters in bacterial species, and they usually are capable of extruding a broad range of structurally unrelated compounds (18).Multidrug efflux pumps of the SMR type are made of a transport protein located in the inner membrane (19). The polypeptide chains of SMR efflux pumps, found in the inner membranes of gram-negative bacteria, are 110 amino acid residues in length and contain four transmembrane helices (29). Reports show that 52% of currently sequenced genomes of eubacteria and archaea contain at least one SMR homologue (2). Well-studied examples of SMR efflux pumps include EmrE of Pseudomonas aeruginosa, EbrAB of Bacillus subtilis, SsmE of Serratia marcescens, and EmrE of Escherichia coli, which are involved in resistance to a variety of antimicrobial agents and quaternary ammonium compounds (14, 16, 17, 34).The 3.9-Mb genome of A. baumannii AYE is reported to harbor 46 open reading frames (ORFs) encoding putative efflux pumps of different families (8). The efflux systems functionally characterized so far include AdeABC and AdeIJK (RND type), AbeM (MATE type), and CraA (MFS type) (20, 27). Albeit comparative genomics clearly reveal the existence of several chromosomally borne putative efflux transporters (8, 12), apparently the role of the Acinetobacter SMR efflux pump was never examined. Therefore, the objective of the present study was to investigate the function of one putative SMR efflux pump from a clinical isolate, A. baumannii AC0037.(Part of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008 [8a].)     

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.     

Characterization of pABVA01, a Plasmid Encoding the OXA-24 Carbapenemase from Italian Isolates of Acinetobacter baumannii

Two epidemiologically unrelated carbapenem-resistant Acinetobacter baumannii isolates were investigated as representatives of the first Italian isolates producing the OXA-24 carbapenemase. Both isolates were of European clonal lineage II and carried an identical OXA-24-encoding plasmid, named pABVA01. Comparative analysis revealed that in pABVA01, bla_OXA-24 was part of a DNA module flanked by conserved inverted repeats homologous to XerC/XerD binding sites, which in other Acinetobacter plasmids flank different DNA modules, suggesting mobilization by a novel site-specific recombination mechanism.Acinetobacter baumannii is an opportunistic nosocomial pathogen of increasing clinical relevance (3, 4, 22). The species is naturally resistant to several antimicrobial agents and exhibits a remarkable propensity to acquire new resistances (4, 14, 22). Carbapenems are elective agents for treatment of A. baumannii infections, and the emergence of carbapenem-resistant strains is a matter of increasing clinical concern (22, 24). Acquired class D carbapenemases of the OXA-23, OXA-24 (also named OXA-40), and OXA-58 lineages are playing a major role as determinants of acquired carbapenem resistance in A. baumannii (24).In Italy, production of OXA-58 is the dominant carbapenem resistance mechanism in A. baumannii, and several outbreaks caused by OXA-58-producing strains related to European clonal lineage II have been documented (11, 15, 17), while strains producing OXA-23 and OXA-24 have not been reported. Here we report the characterization of the first Italian isolates of carbapenem-resistant A. baumannii producing the OXA-24 carbapenemase.(Part of this study was presented at the 18th European Congress of Clinical Microbiology and Infectious diseases, Barcelona, Spain, 2008. [M. M. D''Andrea, T. Giani, F. Luzzaro, and G. M. Rossolini, oral communication O300].)     

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.     

Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.Acinetobacter baumannii is an emerging, opportunistic, Gram-negative bacterial pathogen (19). It is associated with a range of nosocomial infections, including bacteremia, pneumonia, meningitis, and urinary tract infections. Outbreaks, especially in intensive care unit settings, have been identified in numerous countries around the world (23). The treatment of these infections is hampered by the rapid rise in prevalence of A. baumannii strains that are resistant to almost all available antibiotics, including β-lactams, fluoroquinolones, tetracyclines, and aminoglycosides (23). In these multidrug-resistant (MDR) strains, colistin (also known as polymyxin E) is often the only remaining treatment (15), although colistin-resistant clinical isolates have already been reported (7, 10, 21). Intriguingly, some A. baumannii isolates have been shown to display heteroresistance to colistin, where an apparently colistin-susceptible strain (based upon the MIC) harbors a small proportion of colistin-resistant cells (9, 16). Under selective pressure both in vitro (33) and in vivo (10), heteroresistant A. baumannii strains can rapidly give rise to strains with high-level colistin resistance.Colistin is a cationic polypeptide antibiotic that is composed of a cyclic decapeptide linked by an α-amide linkage to a fatty acyl chain (15). Its structure differs from that of polymyxin B by only a single amino acid; the two antibiotics demonstrate comparable activities against a range of Gram-negative bacteria (6). Polymyxins are proposed to exert their antibacterial effect on Gram-negative bacteria via a two-step mechanism comprising initial binding to and permeabilization of the outer membrane, followed by destabilization of the cytoplasmic membrane (37). While the exact mechanism of bacterial killing is not clearly defined, a critical first step in the action of polymyxins is the electrostatic interaction between the positively charged peptide and the negatively charged lipid A, the endotoxic component of lipopolysaccharide (LPS) (3). It has been proposed that because polymyxins target the bacterial outer membrane lipid bilayer, resistance against these antimicrobial peptides is rare (3). However, polymyxin-resistant bacteria have been identified, and the characterized mechanisms of resistance generally involve modifications to lipid A that reduce or abolish this initial charge-based interaction with polymyxins. In many Gram-negative bacteria, modifications of lipid A by addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (PEtn) act to reduce the net LPS negative charge, thereby increasing resistance to polymyxins. The expression of the l-Ara4N and PEtn transferases in Escherichia coli and Salmonella enterica is regulated by the two-component regulatory system PmrA/PmrB, which responds to pH, Fe³⁺ and Mg²⁺ concentrations, as well as the presence of polymyxins (26). While the mechanism(s) of polymyxin resistance in A. baumannii is currently unknown, recent work has indicated that mutations in pmrA and pmrB may be linked to colistin resistance (1). Here we show that in A. baumannii type strain ATCC 19606, colistin-resistant variants contain mutations within genes essential for lipid A biosynthesis (either lpxA, lpxC, or lpxD) and that these strains have lost the ability to produce lipid A and therefore LPS. Furthermore, we show that loss of lipid A leading to colistin resistance is observed in other A. baumannii strains, including a colistin-resistant clinical isolate.     

Correlation between Azole Susceptibilities,Genotypes, and ERG11 Mutations in Candida albicans Isolates Associated with Vulvovaginal Candidiasis in China

The relationship between susceptibilities to fluconazole and itraconazole and microsatellite CAI genotypes were examined from a total of 154 Candida albicans isolates (97 isolates causing vulvovaginitis in Chinese women and 6 vaginal isolates and 51 oral cavity isolates from asymptomatic carriers). The two dominant genotypes, CAI 30-45 (45 isolates) and CAI 32-46 (33 isolates), associated with vulvovaginitis showed significantly different azole susceptibility patterns with strong statistical support. CAI 32-46 isolates were usually less susceptible to both fluconazole and itraconazole than CAI 30-45 isolates and than the oral isolates with other diversified CAI genotypes. Remarkably different mutation patterns in the azole target gene ERG11 were correspondingly observed among C. albicans isolates representing different genotypes and sources. Isolates with the same or similar CAI genotypes usually possessed identical or phylogenetically closely related ERG11 sequences. Loss of heterozygosity in ERG11 was observed in all the CAI 32-46 isolates but not in the CAI 30-45 isolates and most of the oral isolates sequenced. Compared with the ERG11 sequence of strain SC5314 ({"type":"entrez-nucleotide","attrs":{"text":"X13296","term_id":"2503","term_text":"X13296"}}X13296), two homozygous missense mutations (G487T and T916C) leading to two amino acid changes (A114S and Y257H) in Erg11p were found in CAI 32-46 isolates. The correlation between azole susceptibility and C. albicans genotype may be of potential therapeutic significance.Vulvovaginal candidiasis (VVC) is a common vaginal infection, affecting up to 75% of women of child-bearing age at least once in their lifetime (7, 21, 22). The most frequent cause of VVC is Candida albicans, which is responsible for 70 to 90% of vulvovaginitis cases. Non-C. albicans species of Candida, predominantly Candida glabrata, are responsible for the remainder of cases (21). On the basis of the severity of symptoms, frequency, and causative agents, VVC is usually classified as either uncomplicated (mild and sporadic) or complicated (recurrent, severe, or caused by non-C. albicans species) (7, 21). Ten to 20% of women suffer complicated VVC in their lifetime (21). When properly diagnosed, uncomplicated VVC may be treated easily and reliably. However, complicated VVC often causes long-term physical and mental discomfort, significant economic burden from treatments, and considerable negative effect on sexual relations (21-23).At present, prolonged suppressive therapy using fluconazole is recommended as the standard management for chronic, recurrent Candida vulvovaginitis (23). Therefore, there is a great concern about the emergence and spread of azole resistance of C. albicans isolates associated with VVC. Indeed, susceptibility testing of VVC-causing isolates has been performed in different countries and regions of the world (1, 2, 4, 5, 6, 13-15, 17, 18, 20, 24). Although relatively high frequencies of fluconazole- and/or itraconazole-resistant C. albicans isolates causing VVC have been observed in a few reports (13, 20, 24), most studies failed to identify any clear correlation between azole susceptibility and VVC association among C. albicans isolates (1, 2, 4, 5, 6, 14, 15, 17, 18).Recently, we compared the genotype distribution patterns among independent C. albicans isolates associated with VVC in Chinese women and those from various extragenital sites by using the polymorphic microsatellite locus CAI (8, 11). The results showed that the CAI genotypes of C. albicans isolates from extragenital sites were highly diversified. In contrast, isolates associated with VVC from unrelated patients were more homogeneous and belonged to only a few genotypes, with two genotypes, CAI 30-45 and CAI 32-46, being the most common. These two dominant genotypes were rarely found among isolates from extragenital sites (11). In addition, the distribution of the dominant genotypes correlated positively with the severity of VVC (8, 11). These results suggested that C. albicans isolates with genotypes CAI 30-45 and CAI 32-46 might be more virulent and/or more resistant to the commonly used azole drugs than those with other genotypes as causative agents of vaginal infection.Antifungal susceptibility testing using the Etest method revealed that the C. albicans isolates causing VVC in Chinese women were generally susceptible to fluconazole, amphotericin B, ketoconazole, and flucytosine; however, 19.1% of the isolates could be interpreted as being resistant to itraconazole in vitro. Interestingly, most of the itraconazole-resistant isolates belonged to a specific genotype (13). Contrary to the report described above, recent susceptibility testing and microsatellite typing of vulvovaginitis-causing Candida isolates from Europe did not find an association between azole resistance and any particular genotype cluster among C. albicans isolates (1). In the present study, fluconazole and itraconazole susceptibilities of the C. albicans isolates with the dominant genotypes CAI 30-45 and CAI 32-46 from VVC patients were compared with those of isolates possessing other minor genotypes and of isolates from the oral cavity by using the standard broth microdilution method. Furthermore, ERG11 (encoding lanosterol-14-α-demethylase, the target of azoles) gene sequences of C. albicans isolates representing different genotypes and sources were determined. The correlation between azole susceptibilities, genotypes, and ERG11 mutations was examined.     

OXA-143, a Novel Carbapenem-Hydrolyzing Class D β-Lactamase in Acinetobacter baumannii

A carbapenem-resistant Acinetobacter baumannii strain was isolated in Brazil in 2004 in which no known carbapenemase gene was detected by PCR. Cloning experiments, followed by expression in Escherichia coli, gave an E. coli recombinant strain expressing a novel carbapenem-hydrolyzing class D β-lactamase (CHDL). OXA-143 showed 88% amino acid sequence identity with OXA-40, 63% identity with OXA-23, and 52% identity with OXA-58. It hydrolyzed penicillins, oxacillin, meropenem, and imipenem but not expanded-spectrum cephalosporins. The bla_OXA-143 gene was located on a ca. 30-kb plasmid. After transformation into reference strain A. baumannii ATCC 19606, it conferred resistance to carbapenems. Analysis of the genetic environment of bla_OXA-143 revealed that it was associated with neither insertion sequences nor integron structures. However, it was bracketed by similar replicase-encoding genes at both ends, suggesting acquisition through a homologous recombination process. This study identified a novel class D β-lactamase involved in carbapenem resistance in A. baumannii. This enzyme is the first member of a novel subgroup of CHDLs whose prevalence remains to be determined.Acinetobacter baumannii is a nosocomial pathogen that is characterized by its innate and acquired antimicrobial resistance (9, 20, 27). Carbapenems were considered to be the most active antimicrobials against A. baumannii. However, carbapenem resistance is rising and is often associated with a multidrug resistance phenotype (20, 25, 27). The main mechanisms of carbapenem resistance in A. baumannii correspond to efflux pumps, porin mutations, and the production of acquired carbapenem-hydrolyzing class D β-lactamases (CHDLs) (26-28). The impact of overexpression of the naturally occurring bla_OXA-51-like gene remains to be determined, although recent data suggest its involvement in acquired resistance (12). Metallo-β-lactamases are still rarely identified in A. baumannii (25).Some of the class D β-lactamases, also named oxacillinases (OXA), are able to weakly hydrolyze carbapenems. The genes that encode these enzymes are often associated with insertion sequences that provide additional promoter sequences, leading to their overexpression and ultimately to carbapenem resistance (26). To date, four main groups of CHDLs have been identified in A. baumannii, the intrinsic chromosomal OXA-51-like enzyme and the acquired OXA-23-like, OXA-40-like, and OXA-58-like enzymes (5). Acquired OXAs can be either chromosome or plasmid encoded (25). Here we report on a novel plasmid-mediated CHDL that could not be detected by any previous PCR techniques.     

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.     

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).     

Synergy Testing by Etest,Microdilution Checkerboard,and Time-Kill Methods for Pan-Drug-Resistant Acinetobacter baumannii

Pan-drug-resistant (PDR) Acinetobacter baumannii is an important nosocomial pathogen that poses therapeutic challenges. Tigecycline alone or in combination with agents such as colestimethate, imipenem, and/or amikacin is being used clinically to treat PDR A. baumannii infections. The purpose of this study was to compare in vitro susceptibility testing by epsilometric (Etest) methods and the checkerboard (CB) method with testing by time-kill analysis. PDR A. baumannii clinical strains representing eight unique pulsed-field gel electrophoresis clones selected from a total of 32 isolates were tested in vitro with tigecycline, colestimethate, imipenem, and amikacin in single- and two-drug combinations by using two different methods of Etest (with a fixed ratio method [method 1] and with the incorporation of the active drug in medium [method 2]) and by using CB. The three-drug combination of imipenem, tigecycline, and amikacin was also tested by CB. These results were compared to time-kill results. Synergy was consistently detected with the imipenem plus colestimethate and tigecycline plus imipenem combinations. The Etest method with active drug incorporated into the agar allowed us to detect synergy even in the presence of the active drug and was more comparable to CB and time-kill tests. Synergy was detected with the three-drug combination of imipenem, tigecycline, and amikacin by both CB and time-kill methods among several tested clones. These findings indicate the utility of synergy testing to predict activity of specific antibiotic combinations against PDR A. baumannii.In recent years Acinetobacter baumannii, an aerobic, Gram-negative coccobacillus, has emerged as an important nosocomial pathogen due to multiple drug resistance mechanisms, and it can be an extremely difficult microorganism for the clinician to treat (3, 8, 20, 22). It causes a variety of infections that include pneumonia, wound, urinary tract, bloodstream, and intra-abdominal infections (3, 8). We had experienced an increased number of cases of A. baumannii with resistant or intermediate susceptibility patterns to carbapenems over a 2-year time frame at our medical center. Growing numbers of isolates locally, nationally, and internationally have shown resistance to antibiotics such as the carbapenems, which previously had excellent activity in vitro and clinically. Our annual medical center antibiograms and a review of the literature (1, 9, 10, 12, 20, 22, 23, 25, 26) support this. Nontraditional agents, such as colestimethate (polymyxin E) and polymyxin B, despite the associated high toxicities, are being used to treat patients infected with pan-drug-resistant (PDR) A. baumannii (with pan-drug resistance defined as resistance to all routinely tested antimicrobials including carbapenems). Antibiotic resistance has also developed among some strains during treatment with these agents (10, 20). Drug treatment with newer antimicrobials or antimicrobial combinations has become increasingly important to eradicate these infections.Tigecycline was approved by the Food and Drug Administration in June 2005 for treatment of complicated skin and skin structure infections and complicated intra-abdominal infections caused by susceptible strains of bacteria. This glycylcycline antimicrobial has shown a broad spectrum of activity both in vitro and in vivo, and its spectrum includes A. baumannii (21).Therapy with tigecycline alone or in combination with other antimicrobials has included colestimethate, imipenem, amikacin, and ampicillin-sulbactam. In vitro susceptibility data supporting or negating these antibiotics in combination are lacking; there are limited data on whether these combinations act synergistically, additively, or antagonistically. The purpose of this study was to determine combinations of agents that reveal in vitro antimicrobial synergy by two different Etest methods (fixed ratio method [method 1] and with the incorporation of the active drug in medium [method 2]) and the broth microdilution checkerboard (CB) method and to compare these results with time-kill analysis results.     

Alterations in Two-Component Regulatory Systems of phoPQ and pmrAB Are Associated with Polymyxin B Resistance in Clinical Isolates of Pseudomonas aeruginosa

Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 μg/ml polymyxin B (MIC, 8 μg/ml), whereas P. aeruginosa PAO1 grew with 0.25 μg/ml polymyxin B (MIC, 0.5 μg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 μg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 μg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.Pseudomonas aeruginosa is a nosocomial gram-negative opportunistic pathogen that causes a variety of infections (e.g., urinary tract, respiratory, skin, soft tissue, etc.) (3, 12, 18, 19). P. aeruginosa accounts for 11 to 14% of all nosocomial infections and is a major problem for people hospitalized with cancer, cystic fibrosis, or burns (3). Treatment usually involves the use of one or more antibiotics, such as β-lactams, aminoglycosides, or quinolones. Combination therapy usually is recommended for P. aeruginosa infections, as it decreases the risk of antibiotic resistance and enhances the eradication rate. Despite the use of combination therapy, there are numerous reports of the emergence of multidrug-resistant P. aeruginosa. Polymyxins (polymyxin B and colistin) often have been the last resort to treat such isolates (1, 3, 27). However, polymyxin B resistance in multidrug-resistant clinical isolates has been reported (4, 6, 11, 25).The mode of action and the resistance mechanism to polymyxin B has been studied extensively using the reference P. aeruginosa strain PAO1. Polymyxin B is a polycationic lipopeptide antibiotic that interacts with a negatively charged lipid A moiety of the lipopolysaccharide (LPS) of gram-negative bacteria and leads to cell lysis and death (26). Resistance to polymyxin B is caused by the inhibition of the interactions between the antibiotic and the lipid A moiety of the LPS, and the inhibition is based on modifications of lipid A so that it is less negatively charged. LPS modification occurs by the addition of 4-amino-4-deoxy-l-arabinose to lipid A under limiting nutrient conditions, such as 20 μM magnesium or calcium, and is directed by an arnBCADTEF operon modulated by two-component regulatory systems of phoPQ and pmrAB (9, 13, 14, 20). In normal growth conditions, the two-component regulatory systems strictly repress arnBCADTEF, resulting in a phenotype of intrinsic susceptibility to polymyxins (8, 16, 17). Therefore, it is postulated that any interruption of the regulatory systems derepresses arnBCADTEF with resulting resistance to polymyxins. Indeed, in vitro-acquired polymyxin B-resistant P. aeruginosa PAK carried a single-amino-acid substitution in PmrB (20).Although polymyxin-resistant clinical isolates of P. aeruginosa have been reported increasingly worldwide (5, 6, 10, 11, 25), the genetic basis for the resistance in these clinical isolates is unclear. This study aimed to examine the molecular details of polymyxin B resistance among clinical isolates of P. aeruginosa. We characterized five polymyxin B-resistant clinical isolates of P. aeruginosa and found that four isolates carried a single-amino-acid substitution or protein truncation in PhoQ, and one isolate carried dual amino acid substitutions in PmrB. The involvement of the amino acid substitutions or protein truncation in polymyxin B resistance was confirmed by introducing phoPQ or pmrAB containing the amino acid substitutions or protein truncation into P. aeruginosa PAO1 lacking phoPQ or pmrAB.