Haruyoshi Tomita

Potency of carbapenems for the prevention of carbapenem- resistant mutants of Pseudomonas aeruginosa : The high potency of a new carbapenem doripenem

The potencies of the carbapenems; doripenem (DRPM), meropenem (MEPM) and imipenem (IPM) in preventing the emergence of carbapenem-resistant mutants were examined in Pseudomonas aeruginosa strains. The carbapenems predominantly selected carbapenem-resistant mutants or carbapenem mutants with reduced susceptibilities that were specifically resistant to carbapenems and had arisen as a result of the reduced level of expression of the outer membrane protein with a molecular weight of about 48,000 (OprD). The potency of carbapenems in preventing the growth of the mutants differed for DRPM, MEPM and IPM. The isolation frequency of the mutant was examined on agar plates containing each of the carbapenems at a concentration of 1/2 or 1/4 MIC of each carbapenem for that mutant. Mutants were not selected on agar containing DRPM at a frequency of greater than 10−9 per cell per generation, whereas mutants of each strain were selected on agar containing MEPM or IPM at frequencies of 10−7 to 10−9 per cell per generation. The drug concentrations and the drug concentration range for the selective increase of carbapenem resistant mutants in the broth culture containing each carbapenem differed for each carbapenem. DRPM exhibited both the lowest drug concentration and the narrowest range of drug concentration for selection of the carbapenem-resistant mutants. The results shown in this report indicated that DRPM exhibited the greatest ability to prevent the emergence of the mutant.

Possible Connection between a Widely Disseminated Conjugative Gentamicin Resistance (pMG1-Like) Plasmid and the Emergence of Vancomycin Resistance in Enterococcus faecium

ABSTRACT A total of 640 vancomycin-resistant Enterococcus faecium (VRE) isolates, which were obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were used in this study. Of the 640 strains, 611 and 29 were VanA and VanB VRE, respectively, based on PCR analysis. Four hundred ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 μg/ml (MIC) to more than 1,024 μg/ml (MIC). The gentamicin resistance of each of 261 (53%) of the 492 gentamicin-resistant strains was transferred to E. faecium at a frequency of about 10−5 to 10−6 per donor cell in broth mating. More than 90% of vancomycin resistances of the 261 strains cotransferred with the gentamicin resistances to E. faecium strains by filter mating. The conjugative gentamicin resistance plasmids were identified and were classified into five types (A through E) with respect to their EcoRI restriction profiles. The transfer frequencies of each type of plasmid between E. faecium strains or Enterococcus faecalis strains were around 10−3 to 10−5 per donor cell or around 10−6 to 10−7 per donor cell, respectively, in broth mating. Type A and type B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The plasmids did not show any homology in Southern hybridization with the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. The EcoRI or NdeI restriction fragments of each type of plasmids hybridized to the conjugative gentamicin resistance plasmid pMG1 (65.1 kb), which was originally isolated from an E. faecium clinical isolate, and transfer efficiently in broth mating.

Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation.

ABSTRACT The mutation frequency for carbapenem resistance in Pseudomonas aeruginosa strains that were selected with carbapenems was enhanced in the presence of subinhibitory concentrations of fluoroquinolones. The mutants showed either a loss of OprD activity or increased mexAB-oprM expression. The highest mutant isolation frequency was obtained by selection with meropenem, while doripenem inhibited mutant growth.

Molecular Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from Mainland China

ABSTRACT Little is known about vancomycin-resistant enterococci in China. Thirteen pulsed-field gel electrophoresis-confirmed heterogeneous VanA-type vancomycin-resistant Enterococcus faecium (VRE) isolates were obtained from five Chinese hospitals from 2001 to 2005. The isolates were typed by multilocus sequence typing into nine different sequence types (STs), including five new STs (ST18, ST25, ST78, ST203, ST320, ST321, ST322, ST323, and ST335). Vancomycin resistance in each isolate was encoded on conjugative plasmids; two of the plasmids, pZB18 (67 kbp) and pZB22 (200 kbp), were highly conjugative and were able to transfer at high frequencies of around 10−4 and 10−7 per donor cell in broth mating, respectively. None of the plasmids identified in these isolates carried traA, which is usually conserved in the pMG1-like highly conjugative plasmid for E. faecium, implying that pZB18 and pZB22 were novel types of a highly conjugative plasmid in enterococci. Thirteen Tn1546-like elements encoding VanA-type VRE on the conjugative plasmids were classified into six types (types I to VI), and most of them contained both IS1216V and IS1542 insertions. The isolates carrying the type II element were predominant. The six type elements were different from that of a VanA-type Enterococcus faecalis strain isolated from Chinese chicken meat. The results suggested that the disseminations of VRE in these areas were by Tn1546-like elements being acquired by the conjugative plasmids and transferred among E. faecium strains.

Nationwide survey shows that methicillin-resistant Staphylococcus aureus strains heterogeneously and intermediately resistant to vancomycin are not disseminated throughout Japanese hospitals

ABSTRACT A total of 6,625 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 278 hospitals throughout Japan were obtained between November and December 1997 and were examined for their sensitivities to vancomycin using Mueller Hinton (MH), brain heart infusion (BHI), agar plates, or the broth microdilution method. A concentrated inoculum of an MRSA strain or the use of highly enriched medium, such as BHI medium, allows an individual cell to grow on agar plates containing a vancomycin concentration greater than the MIC for the parent strain. However, cells of the colonies which grew on BHI agar plates containing the higher vancomycin concentrations did not acquire a level of vancomycin resistance greater than that of the parent strain and were not subpopulations of heterogeneously vancomycin-resistant MRSA. There was no significance in the fact that these colonies grew on the higher concentration of vancomycin: none showed stable resistance to vancomycin at a concentration above the MIC for the parent strain, and no cell from these colonies showed a relationship between the MIC and the ability of these colonies to grow on higher concentrations of vancomycin. The vancomycin MIC was not above 2 μg/ml for any of the cells originating from these colonies. No Mu3-type heterogeneously resistant MRSA strains, which constitutively produce subpopulations from MRSA clinical isolates with intermediate vancomycin resistance at a high frequency, were detected. There was a unipolar distribution of the MICs ranging from 0.25 to 2 μg of vancomycin/ml among the 6,625 MRSA clinical isolates, indicating that there was no Mu50-type intermediately vancomycin-resistant MRSA (MIC, 8 μg/ml by National Committee for Clinical Laboratory Standards criteria) among the clinical isolates, and there was no evidence of dissemination of Mu3-type MRSA heteroresistant to vancomycin.

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)

ABSTRACT Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10−4 per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOBMG family, which is found in pheromone-independent plasmid pHTβ of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.

A pAD1-Encoded Small RNA Molecule, mD, Negatively Regulates Enterococcus faecalis Pheromone Response by Enhancing Transcription Termination

pAD1 is a 60-kb hemolysin-bacteriocin plasmid in Enterococcus faecalis that encodes a conjugative mating response to a peptide sex pheromone, cAD1, secreted by plasmid-free bacteria. The pheromone response is regulated by two proteins: TraE1, which positively regulates all or most conjugative structural genes, and TraA, which negatively regulates traE1. TraA binds to pAD1 DNA at the iad (encoding the inhibitor peptide iAD1) promoter but is released upon binding to imported pheromone. This leads to enhanced transcription through two closely spaced downstream terminators (t1 and t2) into traE1. TraE1 is believed to then upregulate itself from a site located within t2; thus, a small amount of transcription through t1-t2 could lead to overall induction. It is important therefore that the t1-t2 terminators be tightly controlled to keep the response shut down in the absence of pheromone. A small (200-nucleotide) RNA molecule designated mD is encoded just upstream of t1 by a determinant (traD) oriented in the direction opposite to that of transcripts utilizing t1. mD is expressed at high levels in the uninduced state, but it decreases significantly upon induction. Here we present results of genetic studies relating to the activity of t1-t2 and show that mD strongly enhances transcriptional termination at t1. The mD activity is shown to influence transcription well downstream and can affect the determinant for aggregation substance asa1. The phenomenon is specific in that there is no effect of mD on the unrelated pheromone-responding plasmids pPD1 and pCF10.

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

ABSTRACT The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10−3 per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.

Genetic Analysis of Transfer-Related Regions of the Vancomycin Resistance Enterococcus Conjugative Plasmid pHTβ: Identification of oriT and a Putative Relaxase Gene

The pHT plasmids pHTalpha (65.9 kbp), pHTbeta (63.7 kbp), and pHTgamma (66.5 kbp) are highly conjugative pheromone-independent pMG1-like plasmids that carry Tn1546-like transposons encoding vancomycin resistance. pHTbeta is the prototype plasmid, and the pHTalpha and pHTgamma plasmids are derivatives of the insertion into pHTbeta of an IS232-like (2.2 kbp) element and a group II intron (2.8 kbp), respectively. The complete nucleotide sequence of the pHTbeta plasmid was determined and, with the exception of the Tn1546-like insertion (10,851 bp), was found to be 52,890 bp. Sixty-one open reading frames (ORFs) having the same transcript orientation were identified. A homology search revealed that 22 of the pHTbeta (pHT) plasmid ORFs showed similarities to the ORFs identified on the pXO2 plasmid (96.2 kbp), which is the virulence plasmid essential for capsule formation by Bacillus anthracis; however, the functions of most of the ORFs remain unknown. Most other ORFs did not show any significant homology to reported genes for which functions have been analyzed. To investigate the highly efficient transfer mechanism of the pHT plasmid, mutations with 174 unique insertions of transposon Tn917-lac insertion mutants of pHTbeta were obtained. Of the 174 derivatives, 92 showed decrease or loss in transfer frequency, and 74 showed normal transfer frequency and LacZ expression. Eight derivatives showed normal transfer and no LacZ expression. Inserts within the 174 derivatives were mapped to 124 different sites on pHTbeta. The Tn917-lac insertions which resulted in altered transfer frequency mapped to three separate regions designated I, II, and III, which were separated by segments in which insertions of Tn917-lac did not affect transfer. There was no region homologous to the previously reported oriT sequences in the pHT plasmid. The oriT was cloned by selection for the ability to mobilize the vector plasmid pAM401. The oriT region resided in a noncoding region (192 bp) between ORF31 and ORF32 and contained three direct repeat sequences and two inverted repeat sequences. ORF34, encoding a 506-amino-acid protein which was located downstream of the oriT region, contains the three conserved motifs (I to III) of the DNA relaxase/nickase of mobile plasmids. The transfer abilities of the Tn917-lac-insertion mutants of ORF34 or a mutant of ORF34 with an in-frame motif III deletion were completely abolished. The sequence of the oriT region and the deduced relaxase/nickase protein of ORF34 showed no significant similarity to the oriT and relaxase/nickase of other conjugative plasmids, respectively. The putative relaxase/nickase protein of ORF34 could be classified as a new member of the MOB(MG) family.

Interference of bacterial cell-to-cell communication: a new concept of antimicrobial chemotherapy breaks antibiotic resistance

Bacteria use a cell-to-cell communication activity termed “quorum sensing” to coordinate group behaviors in a cell density dependent manner. Quorum sensing influences the expression profile of diverse genes, including antibiotic tolerance and virulence determinants, via specific chemical compounds called “autoinducers”. During quorum sensing, Gram-negative bacteria typically use an acylated homoserine lactone (AHL) called autoinducer 1. Since the first discovery of quorum sensing in a marine bacterium, it has been recognized that more than 100 species possess this mechanism of cell-to-cell communication. In addition to being of interest from a biological standpoint, quorum sensing is a potential target for antimicrobial chemotherapy. This unique concept of antimicrobial control relies on reducing the burden of virulence rather than killing the bacteria. It is believed that this approach will not only suppress the development of antibiotic resistance, but will also improve the treatment of refractory infections triggered by multi-drug resistant pathogens. In this paper, we review and track recent progress in studies on AHL inhibitors/modulators from a biological standpoint. It has been discovered that both natural and synthetic compounds can disrupt quorum sensing by a variety of means, such as jamming signal transduction, inhibition of signal production and break-down and trapping of signal compounds. We also focus on the regulatory elements that attenuate quorum sensing activities and discuss their unique properties. Understanding the biological roles of regulatory elements might be useful in developing inhibitor applications and understanding how quorum sensing is controlled.