Modulation of Protein A Formation in Staphylococcus aureus by Genetic Determinants for Methicillin Resistance

Many methicillin-resistant (Mec^r) strains of Staphylococcus aureus either produce no protein A or secrete it extracellularly (S. Winblad and C. Ericson, Acta Pathol. Microbiol. Scand. Sect. B 81:150–156, 1973). We found that methicillin resistance and protein A production were apparently lost coordinately from the natively Mec^r strain A676. Restoration of the genetic determinant for methicillin resistance (mec) by transduction or transformation restored protein A production. In two other Mec^r strains, loss of mec was accompanied by marked reduction in protein A formation. Genetic transfer of mec to derivatives of S. aureus 8325 affected protein A formation differently with different mec determinants. Those derived from strain A676 and two other Mec^r strains reduced the scanty amount of protein A produced by strain 8325 to even lower or undetectable levels, whereas mec from two more Mec^r strains increased its protein A content. This “mec-effect,” i.e., stimulation or inhibition of protein A formation dependent on the combination of host strain and mec determinant, was reduced in methicillin-susceptible (Mec^s) mutants produced by ethyl methane sulfonate treatment of Mec^r strains. The mec-effect reappeared in spontaneous revertants to methicillin resistance. Phenotypic reduction of methicillin resistance in Mec^r strains grown at 44°C was accompanied by reduction of the mec-effect on protein A, but it had no effect on protein A formation in Mec^s strains. Two independent mutants of strain 8325 produced large amounts of protein A at rates that were unaffected by growth at 44°C or by the introduction of mec determinants.     

Molecular Epidemiology of Antimicrobial-Resistant Commensal Escherichia coli Strains in a Cohort of Newborn Calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp^r) and nalidixic acid-resistant (Nal^r) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp^r population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal^r population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp^r genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp^r genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp^r isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan^r resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5⁺ from Schizosaccharomyces pombe and the dominant resistance marker ble^r from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP–marker gene–loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble^r as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.     

Contraselectable Streptomycin Susceptibility Determinant for Genetic Manipulation and Analysis of Helicobacter pylori

Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str^r allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str^r] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm^r] and Str^s) were each transformed with wild-type genomic DNAs, and Str^r derivatives were selected. The desired Erm^s Str^r isolates were obtained at frequencies that ranged from 17 to 96% among Str^r transformants, with the Erm^s yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.     

Point Mutations Associated with Organophosphate and Carbamate Resistance in Chinese Strains of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Acetylcholinesterase resistance has been well documented in many insects, including several mosquito species. We tested the resistance of five wild, Chinese strains of the mosquito Culex pipiens quinquefasciatus to two kinds of pesticides, dichlorvos and propoxur. An acetylcholinesterase gene (ace1) was cloned and sequenced from a pooled sample of mosquitoes from these five strains and the amino acids of five positions were found to vary (V185M, G247S, A328S, A391T, and T682A). Analysis of the correlation between mutation frequencies and resistance levels (LC₅₀) suggests that two point mutations, G247S (r² = 0.732, P = 0.065) and A328S (r² = 0.891, P = 0.016), are associated with resistance to propoxur but not to dichlorvos. Although the V185M mutation was not associated with either dichlorvos or propoxur resistance, its RS genotype frequency was correlated with propoxur resistance (r² = 0.815, P = 0.036). And the HWE test showed the A328S mutation is linked with V185M, also with G247S mutation. This suggested that these three mutations may contribute synergistically to propoxur resistance. The T682A mutation was negatively correlated with propoxur (r² = 0.788, P = 0.045) resistance. Knowledge of these mutations may help design strategies for managing pesticide resistance in wild mosquito populations.     

Closely related plasmids from Staphylococcus aureus and soil bacilli

Antibiotic resistance plasmids from staphylococci and soil bacilli have been isolated and compared. A tetracycline resistance (Tc^r) plasmid, indistinguishable from pT181, which is typical of Tc^r plasmids that are widely dispersed among human clinical isolates of S. aureus, has been found also in bovine mastitis isolates. This plasmid, however, shows no detectable homology to a family of related Tc^r plasmids, typified by pBC16, that is widely dispersed among aerobic spore-forming bacilli. However, and rather unexpectedly, pBC16 is highly homologous to and incompatible with pUB110, an S. aureus plasmid specifying kanamycin resistance. The two plasmids are homologous except for the region occupied by their resistance determinants, which has the appearance of a heterologous substitution. These results suggest the occurrence of natural plasmid transfer between staphylococci and soil bacilli.     

Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis, gradual dose-dependent single exposure pediocin-resistant (Ped^r) mutants of E. faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Ped^r mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in Ped^r E. faecalis. Decreased level of branched chain fatty acids in Ped^r mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Ped^r mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Ped^r mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC₅₀ of CAMPs for wild-type and Ped^r mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs.     

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs^r) cassette linked to a bacterial tetracycline resistance (tet^r) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs^r-tet^r cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.     

The Connection between Persistent,Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis

The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC^r) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC^r. Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC^r isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Intraspecies and Interspecies Transformation Reactions in Pneumococcus and Streptococcus

The efficiency of transformation of pneumococcus and a strain of viridans streptococcus (strain D) to streptomycin resistance is influenced by the species in which the mutation to resistance occurred, as well as by the species in which the mutated gene has been replicated. Pneumococcus and streptococcus strain D transform in higher frequency with DNA that has been replicated in bacteria of the same species than with DNA from the heterologous species. However, the difference between the frequencies of interspecific and intraspecific transformation is much greater with pneumococcus as receptor than with streptococcus. In addition pneumococcus transforms in higher frequency with wholly homologous (pneumococcal) DNA than with DNA from pneumococci that have replicated the streptococcal Sm^r gene. Pneumococcus is transformed in lower frequency by wholly heterologous (streptococcal) DNA than by DNA from streptococci that have replicated the pneumococcal Sm^r gene. Streptococcus behaves similarly in that wholly homologous (streptococcal) DNA transforms it more efficiently than when the transforming fragment contains a pneumococcal moiety. Streptococcus is transformed in the same or lower frequency by wholly heterologous (pneumococcal) DNA than by DNA from pneumococci that have replicated the streptococcal Sm^r gene. When erythromycin resistance was used as genetic marker instead of streptomycin resistance, similar results were found.     

Organization of the Rudimentary Wing Locus in DROSOPHILA MELANOGASTER. I. the Isolation and Partial Characterization of Mutants Induced with Ethyl Methanesulfonate, Icr-170 and X Rays

New rudimentary (r) mutants have been isolated following mutagenesis with ethyl methanesulfonate (r^LE), ICR–170 (r^LI) and X rays (r^LX). From wing phenotype measurements on homoallelic females, it has been shown that the r^LE mutant series includes several leaky alleles, as well as alleles that produce moderate and strong r phenotypes. All of the tested r^LI alleles yielded strong r phenotypes in homoallelic females, whereas the r^LX series was found to include both moderate and strong alleles. Based on allele complementation for the wing phenotype, it was found that all three mutant series include both complementing and noncomplementing alleles, but the relative frequencies of these two types of alleles differ considerably among the three series. Complementing alleles comprise most of the r^LE mutant series (19 of 25) and almost one-half of the r^LX series (five of 12), while only one of 16 r^LI mutants is a complementing allele. Data from enzyme assays of mutants mostly support the direct correlation of genetic complementation units with the activities of the first three enzymes in the de novo pyrimidine biosynthetic pathway. All of these findings are discussed in light of evidence that these three enzymes are contained within a trienzyme complex in animals. We conclude that the available genetic evidence supports the contention that the trienzyme complex is encoded by a single mRNA.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.     

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc^r) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc^r genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc^r genes was quantified by real-time quantitative PCR. To confirm the Tc^r gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc^r genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc^r genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.     

Transduction versus transformation of methicillin resistance inStaphylococcus aureus

Requirements for the transformation of methicillin resistance (Mec^r) inStaphylococcus aureus were found to differ from those of transduction in three significant respects. Production of -lactamase by the recipient strain is not a stringent requirement for Mec^r transformation as it is for transduction althoughbla ⁺ recipients exhibited a higher transformation frequency in the absence of added NaCl. Incorporation of NaCl into the methicillin-selective media markedly inhibited Mec^r transduction but increased the transformation frequency of plasmid-free recipients to the level obtained withbla ⁺ recipients. Four separately maintained clones of strain 8325-4 exhibited marked variation in recipient effectiveness for Mec^r transduction but were equally effective as recipients in transformation.     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.