Lapatinib Antagonizes Multidrug Resistance–Associated Protein 1–Mediated Multidrug Resistance by Inhibiting Its Transport Function

Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance–associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.     

Do We Need to Detect Isoniazid Resistance in Addition to Rifampicin Resistance in Diagnostic Tests for Tuberculosis?

Background

Multidrug-resistant tuberculosis (MDR-TB) is resistant to both rifampicin (RIF) and isoniazid (INH). Whereas many TB diagnostics detect RIF-resistance, few detect INH-monoresistance, which is common and may increase risk of acquired MDR-TB. Whether inclusion of INH-resistance in a first-line rapid test for TB would have an important impact on MDR-TB rates remains uncertain.

Methods

We developed a transmission model to evaluate three tests in a population similar to that of India: a rapid molecular test for TB, the same test plus RIF-resistance detection (“TB+RIF”), and detection of RIF and INH-resistance (“TB+RIF/INH”). Our primary outcome was the prevalence of INH-resistant and MDR-TB at ten years.

Results

Compared to the TB test alone and assuming treatment of all diagnosed MDR cases, the TB+RIF test reduced the prevalence of MDR-TB among all TB cases from 5.5% to 3.8% (30.6% reduction, 95% uncertainty range, UR: 17–54%). Despite using liberal assumptions about the impact of INH-monoresistance on treatment outcomes and MDR-TB acquisition, expansion from TB+RIF to TB+RIF/INH lowered this prevalence only from 3.8% to 3.6% further (4% reduction, 95% UR: 3–7%) and INH-monoresistant TB from 15.8% to 15.1% (4% reduction, 95% UR: (-8)-19%).

Conclusion

When added to a rapid test for TB plus RIF-resistance, detection of INH-resistance has minimal impact on transmission of TB, MDR-TB, and INH-monoresistant TB.     

Tracking Acquired Antibiotic Resistance in Commensal Bacteria of Galápagos Land Iguanas: No Man,No Resistance

Background

Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago.

Methodology/Principal Findings

Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact.

Conclusions/Significance

Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Resistance of Clostridium perfringens Type A Spores to γ-Radiation

The radiation resistance of the spores of a classical strain and of an atypical, heat-resistant strain of Clostridium perfringens was determined. Spores were produced in Ellner's and in a Trypticase broth medium. Approximately 10⁶ viable spores per milliliter were suspended in 0.06 m phosphate buffer and irradiated with γ rays from cobalt-60; the survivors were counted in Tryptone-yeast extract-agar by the Prickett-tube technique. Radiation D values for spores of the atypical strain in phosphate buffer and in cooked-meat broth were 0.23 and 0.30 Mrad, respectively, and the D value of the classical strain was 0.25 Mrad in phosphate buffer. Spores of the classical and atypical strains of C. perfringens type A are characterized by differences in heat resistance; yet, all strains tested demonstrated similar radiation resistance. Also, the spores were more resistant to ionizing radiation in cooked-meat broth than in phosphate buffer.     

Implication of Porins in β-Lactam Resistance of Providencia stuartii

An integrative approach combining biophysical and microbiological methods was used to characterize the antibiotic translocation through the outer membrane of Providencia stuartii. Two novel members of the General Bacterial Porin family of Enterobacteriaceae, named OmpPst1 and OmpPst2, were identified in P. stuartii. In the presence of ertapenem (ERT), cefepime (FEP), and cefoxitin (FOX) in growth media, several resistant derivatives of P. stuartii ATCC 29914 showed OmpPst1-deficiency. These porin-deficient strains showed significant decrease of susceptibility to β-lactam antibiotics. OmpPst1 and OmpPst2 were purified to homogeneity and reconstituted into planar lipid bilayers to study their biophysical characteristics and their interactions with β-lactam molecules. Determination of β-lactam translocation through OmpPst1 and OmpPst2 indicated that the strength of interaction decreased in the order of ertapenem ≫ cefepime > cefoxitin. Moreover, the translocation of these antibiotics through OmpPst1 was more efficient than through OmpPst2. Heterologous expression of OmpPst1 in the porin-deficient E. coli strain BL21(DE3)omp8 was associated with a higher antibiotic susceptibility of the E. coli cells to β-lactams compared with expression of OmpPst2. All our data enlighten the involvement of porins in the resistance of P. stuartii to β-lactam antibiotics.     

The Path to Insulin Resistance: Paved with Ceramides?

Obesity-associated, system-wide elevations in free fatty acids, tumor necrosis factor alpha, and glucocorticoids increase intracellular lipid metabolites and promote insulin resistance. In this issue, Holland et al. (2007) provide pharmacological and genetic evidence that ceramide plays a key role in the development of insulin resistance induced by these factors.     

Introduction of Resistance to Herbicide Basta® in Savoy Cabbage

Resistance to herbicide Basta® was introduced into pure inbred lines of Savoy cabbage (Brassica oleracea L. var. sabauda) by cocultivation of cotyledon and hypocotyl explants with Agrobacterium tumefaciens strains AGL1/pDM805 and LBA4404/pGKB5 (LB5-1). Shoot regeneration occurred on Murashige and Skoog medium supplemented with 1 mg dm^–3 6-benzyladenine and 0.5 mg dm^–3 indole-3-butyric acid at 42.3 % and 71.4 % of hypocotyl explants treated with AGL1/pDM805 and LB5-1, respectively. Putative transformants that survived selection on 10 mg dm^–3 phosphinothricin (L-PPT) supplemented medium were confirmed by GUS assay and PCR analysis. The transformation rate was 58 % with AGL1/ pDM805 and 25 % with LB5-1. Rooted plantlets were acclimated and then again screened for Basta®-resistance by spraying with 15 – 60 mg dm^–3 L-PPT. Surviving plants were selfed and Basta®-resistance was demonstrated in T₁ progeny.     

Antibiotic Resistance Among Anaerobes: What Does it Mean?

Antibiotic resistance among anaerobes is increasing, with significant resistance to clindamycin, cephalosporins, cephamycins, and penicillins noted at community hospitals and major medical centers. A total of 615 anaerobes isolated from various Chicago area hospitals in 1996 were tested against 13 antibiotics, and the resistance patterns compared with similar data from 1991. For the Bacteroides fragilis group anaerobes, the most effective antibiotics were the β-lactam/β-lactamase inhibitor combination agents, carbapenems, trovafloxacin and metronidazole. High levels of resistance to clindamycin, piperacillin, cefoxitin and ceftizoxime were seen 1996. For non- B. fragilis group anaerobes, resistance was mush lower, and was notable only in Clostridium spp. (clindamycin and cephamycins) and Prevotella spp. (clindamycin and piperacillin). Despite the prevalence of antibiotic resistance among anaerobes, the frequency of antimicrobial susceptibility testing of anaerobes is declining. There are a number of factors that account for this decline, including a general reduction in funding of hospital clinical laboratories, a concomitant loss of expertise at these institutions, a lack of automated testing for anaerobes, and a failure to consider resistance as important to clinicians. The case for increased susceptibility testing is built upon the changing patterns of resistance such as those reported in this paper, the identification and transfer of genetic determinants corresponding to antibiotic resistance, as well as the correlation of resistance and clinical outcome.     

β-Lactam Resistance in Salmonella Strains Isolated from Retail Meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006

Ampicillin-resistant (Amp^r) Salmonella enterica isolates (n = 344) representing 32 serotypes isolated from retail meats from 2002 to 2006 were tested for susceptibility to 21 other antimicrobial agents and screened for the presence of five beta-lactamase gene families (bla_CMY, bla_TEM, bla_SHV, bla_OXA, and bla_CTX-M) and class 1 integrons. Among the Amp^r isolates, 66.9% were resistant to five or more antimicrobials and 4.9% were resistant to 10 or more antimicrobials. Coresistance to other β-lactams was noted for amoxicillin-clavulanic acid (55.5%), ceftiofur (50%), cefoxitin (50%), and ceftazidime (24.7%), whereas less than 5% of isolates were resistant to piperacillin-tazobactam (4.9%), cefotaxime (3.5%), ceftriaxone (2%), and aztreonam (1.2%). All isolates were susceptible to cefepime, imipenem, and cefquinome. No Salmonella producing extended-spectrum beta-lactamases was found in this study. Approximately 7% of the isolates displayed a typical multidrug-resistant (MDR)-AmpC phenotype, with resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, plus resistance to amoxicillin-clavulanic acid, cefoxitin, and ceftiofur and with decreased susceptibility to ceftriaxone (MIC ≥ 4 μg/ml). Pulsed-field gel electrophoresis results showed that several MDR clones were geographically dispersed in different types of meats throughout the five sampling years. Additionally, 50% of the isolates contained bla_CMY, 47% carried bla_TEM-1, and 2.6% carried both genes. Only 15% of the isolates harbored class I integrons carrying various combinations of aadA, aadB, and dfrA gene cassettes. The bla_CMY, bla_TEM, and class 1 integrons were transferable through conjugation and/or transformation. Our findings indicate that a varied spectrum of coresistance traits is present in Amp^r Salmonella strains in the meat supply of the United States, with a continued predominance of bla_CMY and bla_TEM genes in β-lactam-resistant isolates.Nontyphoid Salmonella enterica is one of the most important food-borne pathogens and represents a significant public health hazard worldwide. It is estimated that 1.4 million people in the United States are infected with non-Typhi Salmonella annually, resulting in 15,000 hospitalizations and more than 400 deaths (28). Salmonella infections in humans often result from the ingestion of contaminated foods, such as poultry, beef, pork, eggs, milk, seafood, and produce (10). Salmonellosis following direct contact with animals and dog treats has also been reported (3, 6, 7). Human salmonellosis usually results in a self-limiting diarrhea that does not require antimicrobial therapy. However, in severe cases of enteritis and systemic infections, fluoroquinolones and extended-spectrum cephalosporins such as ceftriaxone (AXO) are used as first-line therapeutics (12, 27).Multidrug-resistant (MDR) Salmonella strains have been detected in many serotypes, such as S. enterica serotype Typhimurium (9, 26), S. enterica serotype Agona, S. enterica serotype Anatum, S. enterica serotype Choleraesuis, S. enterica serotype Dublin, S. enterica serotype Heidelberg, S. enterica serotype Kentucky, S. enterica serotype Newport, S. enterica serotype Schwarzengrund, S. enterica serotype Senftenberg, and S. enterica serotype Uganda, among others (14, 33, 35) (http://internet-dev/cvm/2005NARMSExeRpt.htm). The most common MDR pattern, which first emerged in S. Typhimurium, has been a pattern of resistance to ampicillin (AMP), chloramphenicol (CHL), streptomycin (STR), sulfonamides, and tetracycline (TET) (ACSSuT). More recently, strains exhibiting the ACSSuT pattern also have acquired MDR plasmids carrying the bla_CMY gene and others (30) that can spread readily to different members of the Enterobacteriaceae. The strains demonstrate extensive resistances, which, in addition to the ACSSuT phenotype, may include resistance to amoxicillin-clavulanic acid (AUG), cefoxitin (FOX), and ceftiofur (TIO) and decreased susceptibility to AXO (MIC ≥ 4 μg/ml). TIO is a third-generation cephalosporin that was approved for use in animals in 1998. Tio^r Salmonella isolates often show resistance or decreased susceptibility to AXO (also a third-generation cephalosporin used to treat human infections). Some strains may also display resistance to gentamicin (GEN), kanamycin (KAN), and trimethoprim-sulfamethoxazole ([SMX] COT) as well as resistance to disinfectants and heavy metals. Resistance to third-generation cephalosporins in Salmonella strains is of interest because these are the drugs of choice for treating salmonellosis in children, where fluoroquinolones are contraindicated (13).To date, more than 340 beta-lactamases have been described (11). The most common genes, such as bla_TEM, bla_SHV, bla_CTX-M, bla_OXA, bla_PER, bla_PSE, and bla_CMY, have been detected in Salmonella, with the prevalence of these genes varying by region (32). Extended-spectrum beta-lactamases (ESBLs) are less prevalent in Salmonella strains than in other gram-negative bacteria such as Klebsiella, Escherichia coli, and Proteus. The ESBLs are β-lactamases capable of conferring bacterial resistance to the penicillins; to first-, second-, and third-generation cephalosporins; and to aztreonam (ATM) (but not to the cephamycins or carbapenems) by hydrolysis of these antibiotics, which are inhibited by β-lactamase inhibitors such as clavulanic acid (21). Most ESBL-carrying Salmonella strains have been reported in Latin America, the Western Pacific, and Europe (32), with only a few reports from North America. In the United States the first case was reported in 1994, when bla_CTX-5 was detected in an S. Typhimurium var. Copenhagen strain from an infant adopted from Russia (25). Additional ESBL Salmonella strains have been reported recently, one from a horse (bla_SHV-12) and another from a 3-month-old child (bla_CTX-M-5) (23, 25). Carbapenem resistance in Salmonella is also rare in the United States but has been detected in S. enterica serotype Cubana associated with a plasmid-mediated bla_KPC-2 gene (18). In contrast to the low prevalence of ESBL-carrying Salmonella strains in the United States, AmpC resistance mediated by bla_CMY has been emerging in both humans and food animals. The bla_CMY encodes a cephalomycinase that exhibits extended resistance to many beta-lactams, including first-, second-, and third-generation cephalosporins (36).The objectives of this study were to determine the genetic basis of beta-lactam resistance and to examine the extent of coresistance to other antimicrobials among 344 Amp^r Salmonella isolates obtained from retail meats. We screened for the presence of five beta-lactam resistance gene families (bla_CMY, bla_TEM, bla_SHV, bla_OXA, and bla_CTX-M) and the presence of class 1 integrons. The range of resistance phenotypes borne on plasmids was examined by filter mating and electroporation, and all isolates were characterized for genetic relatedness using pulsed-field gel electrophoresis (PFGE).     

Non-Invasive Pneumococcal Pneumonia in Portugal—Serotype Distribution and Antimicrobial Resistance

There is limited information on the serotypes causing non-invasive pneumococcal pneumonia (NIPP). Our aim was to characterize pneumococci causing NIPP in adults to determine recent changes in serotype prevalence, the potential coverage of pneumococcal vaccines and changes in antimicrobial resistance. Serotypes and antimicrobial susceptibility profiles of a sample of 1300 isolates recovered from adult patients (≥18 yrs) between 1999 and 2011 (13 years) were determined. Serotype 3 was the most frequent cause of NIPP accounting for 18% of the isolates. The other most common serotypes were 11A (7%), 19F (7%), 19A (5%), 14 (4%), 22F (4%), 23F (4%) and 9N (4%). Between 1999 and 2011, there were significant changes in the proportion of isolates expressing vaccine serotypes, with a steady decline of the serotypes included in the 7-valent conjugate vaccine from 31% (1999–2003) to 11% (2011) (P<0.001). Taking together the most recent study years (2009–2011), the potential coverage of the 13-valent conjugate vaccine was 44% and of the 23-valent polysaccharide vaccine was 66%. While erythromycin resistance increased from 8% in 1999–2003 to 18% in 2011 (P<0.001), no significant trend was identified for penicillin non-susceptibility, which had an average value of 18.5%. The serotype distribution found in this study for NIPP was very different from the one previously described for IPD, with only two serotypes in common to the ones responsible for half of each presentation in 2009–2011 – serotypes 3 and 19A. In spite of these differences, the overall prevalence of resistant isolates was similar in NIPP and in IPD.     

Biofilms and Biocides: Are there Implications for Antibiotic Resistance?

Considerable controversy surrounds the use of biocides in an ever increasing range of consumer products and the possibility that their indiscriminate use might reduce biocide effectiveness and alter susceptibilities towards antibiotics. These concerns have been based largely on the isolation of resistant mutants from in vitro monoculture experiments. To date, however the emergence of biocide-resistant strains in-vivo has not been reported and a number of environmental survey studies have failed to associate biocide use with antibiotic resistance. This article gives an overview of the issues as they currently stand and reviews data generated in our laboratory over the last five years where we have used laboratory microcosms of the environment and oral cavity to better understand the possible effects of real-life biocide exposure of these high risk ecosystems. In general, whilst biocide susceptibility changes can be demonstrated in pure culture, especially for E. coli towards triclosan, it has not been possible to reproduce these effects during chronic, sublethal dosing of complex communities. We conclude from this review that whilst the incorporation of antibacterial agents into a widening sphere of personal products may not overtly impact on the patterns of microbial susceptibility observed in the environment, the precautionary principle suggests that the use of biocides should be limited to applications where clear hygienic benefits can be demonstrated.