MgrA activates expression of capsule genes, but not the α-toxin gene in experimental Staphylococcus aureus endocarditis.

BACKGROUND: Staphylococcus aureus produces numerous virulence factors but little is known about their in vivo regulation during an infection. METHODS: The production of capsule and α-toxin, and the expression of their respective genes, cap5 and hla, were analyzed by comparing CYL11481 (derivative of Newman) and its isogenic regulatory mutants in vitro. The temporal expression of cap5 and hla and the regulatory genes in vivo was carried out using a rat infective endocarditis model. RESULTS: In vitro analyses showed that capsule was positively regulated by MgrA, Agr, Sae, ArlR, and ClpC, and negatively by CodY and SbcDC. The α-toxin was positively regulated by MgrA, Agr, Sae, ArlR, and SbcDC but negatively by ClpC and CodY. In vivo analyses showed that cap5 expression correlated best with mgrA expression, whereas hla expression correlated best with sae expression. Mutation in mgrA drastically reduced cap5 expression in vivo. CONCLUSIONS: Our results suggest that, in vitro, Agr is the most important regulator for capsule and α-toxin production, as well as for cap5 transcription, but SaeR is the most critical for hla transcription. However, in vivo, MgrA is the major transcriptional regulator of capsule, but not α-toxin, whereas saeR expression correlates best with hla expression.

Nosocomial outbreak of genetically related IMP-1 beta-lactamase-producing Klebsiella pneumoniae in a general hospital in Japan.

Gram-negative bacteria with acquired metallo-beta-lactamase (MBL) resistance are being increasingly described worldwide. Here we report the first case of an outbreak by a cluster of genetically related strains of Klebsiella pneumoniae producing the IMP-1 MBL. Six isolates of K. pneumoniae with a ceftazidime minimum inhibitory concentration >/=64 microg/mL were collected between February 2003 and June 2004 in Hanyu General Hospital, Saitama, Japan. These isolates were analysed to establish the mechanism of resistance. The zone of inhibition of these isolates using ceftazidime or imipenem disks on Mueller-Hinton agar containing dipicolinic acid was much larger than on Mueller-Hinton agar without dipicolinic acid. Polymerase chain reaction and DNA sequencing confirmed that the isolates contained bla(IMP-1) as well as intI1 as a class I integrase gene. Pulsed-field gel electrophoresis was performed, showing that five of the six isolates were related. This outbreak was controlled by restrained and careful use of antibiotics as well as strict hygiene practices.

Evaluation of antimicrobial activity of beta-lactam antibiotics by Etest against clinical isolates from 100 medical centers in Japan (2004).

This antimicrobial resistance surveillance study was performed in 100 medical centers. The susceptibility of 9347 strains including Escherichia coli (997 strains), Klebsiella spp. (997 strains), Enterobacter spp. (988 strains), Citrobacter spp. (834 strains), indole-positive Proteae spp. (855 strains), Serratia spp. (925 strains), Acinetobacter spp. (902 strains), Pseudomonas aeruginosa (996 strains), oxacillin-susceptible Staphylococcus aureus (992 strains), and coagulase-negative staphylococci (861 strains) to 7 beta-lactam antibiotics, cefepime, cefpirome, ceftazidime, cefoperazone/sulbactam, imipenem and piperacillin (for gram negatives), or oxacillin (for gram positives) was tested. No strain resistant to these beta-lactams except for ceftazidime was found in oxacillin-susceptible S. aureus and coagulase-negative staphylococci. E. coli (16.5%) clinical isolates were resistant to piperacillin, whereas 1.5% or less (cefpirome = 1.5%) was resistant to other beta-lactams. Klebsiella spp. strains were more susceptible to imipenem (99.7%), cefepime (98.4%), and cefpirome (97.3%). Isolates of Enterobacter spp., Citrobacter spp., indole-positive Proteae, and Serratia spp. were susceptible to imipenem, cefepime, and cefpirome, as well. Acinetobacter spp. strains were most susceptible to cefoperazone/sulbactam (0.8% resistance), imipenem (3.2%), ceftazidime (6.0%), and cefepime (7.0%) than other beta-lactam antibiotics tested. Isolates of P. aeruginosa were more susceptible to ceftazidime (9.9% resistance), cefoperazone/sulbactam (14.9%), and cefepime (11.2%) than piperacillin (15.5%), cefpirome (19.1%), and imipenem (19.3%). The percentage of imipenem-resistant P. aeruginosa is around 20% in clinical isolates in Japan.

Identification of biochemically atypical Staphylococcus aureus clinical isolates with three automated identification systems.

Between January and April 2002, a total of 271 strains of Staphylococcus aureus were isolated from clinical specimens at Toho University Omori Hospital, Japan, including 201 (74.2 %) which were identified as meticillin-resistant S. aureus (MRSA). However, 34 (12.5 %) were biochemically atypical, because they did not produce acid on mannitol salt agar or did not agglutinate in Staphaurex testing but were categorized as MRSA by PCR analysis and by antibiotic susceptibility. Three automatic identification systems, AutoScan-4 (Dade Behring), BD Phoenix (Becton Dickinson) and Vitek 2 (bioMérieux), were evaluated by testing these atypical S. aureus isolates. The AutoScan-4 and Phoenix systems identified all 34 isolates as S. aureus. Without additional tests such as Staphaurex, observation of colony pigment and haemolysins on sheep blood agar, Vitek 2 identified only 16 isolates (47.1 %) as S. aureus with good or better confidence levels and misidentified one of the remaining isolates as Staphylococcus chromogenes. This study shows that it is possible to identify these physiologically atypical S. aureus isolates correctly by using the Phoenix and AutoScan-4 fully automatic identification systems.

Metallo-beta-lactamase IMP-1 in Providencia rettgeri from two different hospitals in Japan.

In 2002, 495 indole-positive proteae strains were isolated from patients at 60 hospitals in Japan. Nine indole-positive proteae strains had reduced susceptibility to imipenem (MIC > or = 8 microg ml(-1)) and were identified as Providencia rettgeri by BD Phoenix. Eight of the nine Prov. rettgeri isolates were confirmed as metallo-beta-lactamase producers by the double-disc synergy test. All the metallo-beta-lactamases were classified as IMP-1 by PCR and DNA sequence analysis. These bla(IMP-1) genes were encoded in the integron structure on conjugative plasmids. These plasmids could transfer from Prov. rettgeri clinical isolates to Escherichia coli ML4903 at a frequency between 1.5 x 10(-5) and 5.5 x 10(-7). The eight bla(IMP)-positive strains were isolated from two hospitals, and showed two different PFGE patterns, two different integron structures and two different incompatibility groups, which corresponded to the two hospitals. These results strongly suggest the possibility of nosocomial infections by bla(IMP-1)-producing Prov. rettgeri isolates.

Evaluation of antimicrobial activity of beta-lactam antibiotics using Etest against clinical isolates from 60 medical centres in Japan.

An antimicrobial resistance surveillance study was carried out in 60 medical centres across Japan. Resistance to piperacillin was 10.8% in clinical isolates of Escherichia coli, while 1.3% or fewer isolates were resistant to other beta-lactams. Klebsiella spp. were more susceptible to imipenem, cefepime and cefpirome. Isolates of Enterobacter spp., Citrobacter spp., indole-positive Proteus and Serratia spp. were susceptible to imipenem, cefepime and cefpirome, while Acinetobacter spp. were most susceptible to cefoperazone/sulbactam, imipenem, ceftazidime (5.8% resistance) and cefepime (7.6%). Isolates of Pseudomonas aeruginosa were more susceptible to ceftazidime (12.3% resistance), cefoperazone/sulbactam (12.5%) and cefepime (12.6%) than to piperacillin (15.0%), cefpirome (22.6%) and imipenem (30.8%). The percentage of Japanese imipenem resistant P. aeruginosa clinical isolates was around 30%.

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase.

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome.

BACKGROUND: Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. FINDINGS: We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site. CONCLUSIONS: In this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.

Kinetics study of KPC-3, a plasmid-encoded class A carbapenem-hydrolyzing beta-lactamase.

The kinetic activity of KPC-3, a plasmid-encoded class A carbapenemase, was studied. It hydrolyzed penicillins, cephalosporins, carbapenems, and even sulbactam. The best substrate was cephalothin (k(cat/K)m = 3.48 microM(-1) s(-1)). The efficiency of the enzyme was similar for imipenem and meropenem (k(cat)/K(m), 1.4 and 1.94 microM(-1) s(-1), respectively).

Kinetic properties of four plasmid-mediated AmpC beta-lactamases.

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC beta-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K(m) values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward beta-lactam antibiotics are mainly due to the overproduction of the beta-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded beta-lactamases.

Novel SHV-derived extended-spectrum beta-lactamase, SHV-57, that confers resistance to ceftazidime but not cefazolin.

A new SHV-derived extended-spectrum beta-lactamase, SHV-57, that confers high-level resistance to ceftazidime but not cefotaxime or cefazolin was identified from a national surveillance study conducted in Taiwan in 1998. An Escherichia coli isolate resistant to ampicillin, cephalothin, and ceftazidime but sensitive to cefoxitin, ceftriaxone, cefotaxime, imipenem, and a narrow-spectrum cephem (cefazolin) was isolated from the urine of a patient treated with beta-lactam antibiotics. Resistance to beta-lactams was conjugatively transferred with a plasmid of about 50 kbp. The pI of this enzyme was 8.3. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 861 bases (GenBank accession number AY223863). Kinetic parameters showed that SHV-57 had a poor affinity to cefazolin. The K(m) value toward cefazolin (5.57 x 10(3) muM) was extremely high in comparison to those toward ceftazidime (30.9 muM) and penicillin G (67 muM), indicating its low affinity to cefazolin. Although the K(m) value of the beta-lactamase inhibitor was too high for the study of catalytic activity (k(cat)), indicating the low k(cat) of SHV-57, the SHV-57 carrier was highly susceptible to a beta-lactam-beta-lactamase inhibitor combination. Comparison of the three-dimensional molecular model of SHV-57 with that of the SHV-1 beta-lactamase suggests that the substitution of arginine for leucine-169 in the Omega loop is important for the substrate specificity.

Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a new concern.

Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx(1) was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 microg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 microg of clavulanic acid per ml, the MIC of cefotaxime decreased to < or =0.12 microg/ml, indicating that this strain was an extended-spectrum beta-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 x 10(-6). A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of beta-lactamases. This beta-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.

Clonal diversity of metallo-beta-lactamase-possessing Pseudomonas aeruginosa in geographically diverse regions of Japan.

The aim of this study was to determine the distribution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in Japan and to investigate the molecular characteristics of resistance gene cassettes including the gene encoding this enzyme. A total of 594 nonduplicate strains of P. aeruginosa isolated from 60 hospitals throughout Japan in 2002 were evaluated. This study indicated that although the prevalence of imipenem-resistant P. aeruginosa has not increased compared to that found in previous studies, clonal distribution of the same strain across Japan is evident.

Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis.

CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum beta-lactamase, which differs from the majority of CTX-M-type beta-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime. To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the beta-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed. We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 micro g/ml). Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (k(cat)/K(m), 1.47 x 10(6) s(-1) M(-1)), cefoxitin (k(cat)/K(m), 62.2 s(-1) M(-1)), and aztreonam (k(cat)/K(m), 1.34 x 10(3) s(-1) M(-1)) are good substrates and that imipenem (k(+2)/K, 1.20 x 10(2) s(-1) M(-1)) is a poor substrate. However, CTX-M-18 and CTX-M-19 exhibited too high a K(m) value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (k(cat)). Comparison of the MICs with the catalytic efficiency (k(cat)/K(m)) of these enzymes showed that some beta-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation. Using the previously reported crystal structure of the Toho-1 beta-lactamase, which belongs to the CTX-M-type beta-lactamase group, we have suggested characteristic interactions between the enzymes and the beta-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling. Aminothiazole-bearing beta-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of beta-lactams.

Cefcapene inactivates chromosome-encoded class C beta-lactamases.

The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes of beta-lactamases was evaluated. For the class A beta-lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter ( k(cat)/ Km) values were calculated (3100 - 1.1 x 10(7) M(-1) x s(-1)), because these enzymes have very high Km values for cefpodoxime and cefotaxime. As for class B beta-lactamases L1, IMP-1, and CcrA, in general, similar k(cat)/ Km values were obtained. However, regarding class C beta-lactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds. Cefpodoxime acted as a good substrate for the class C beta-lactamases, except for the enzyme from E. cloacae; its k(cat) and Km values were successfully calculated ( k(cat)/ Km, 1.8 x 10(5) - 1.2 x 10(7) M(-1) x s(-1)). On the other hand, cefcapene acted as a poor substrate or an inactivator for class C beta-lactamases; its k(2)/ K value was successfully calculated (8.7 x 10(5) - 7.0 x 10(6) M(-1) x s(-1)). In addition, k(3) values were determined for beta-lactamases from P. aeruginosa (2.3 x 10(-2) x s(-1)) and C. freundii (2.1 x 10(-1) x s(-1)). Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed. These findings suggest the potential of cephem compounds as inhibitors of class C beta-lactamases.

Rapid pulsed-field gel electrophoresis technique for determination of genetic diversity of Serratia marcescens.

Seventeen Serratia marcescens strains were isolated from patients admitted to a hospital in the Tokyo metropolitan area. In order to study their genetic diversity, pulsed-field gel electrophoresis (PFGE) was performed. Clear bands were not observed by the standard PFGE technique following the instruction manual from the company. To obtain clear results, it was necessary to include some additional steps to the preparation of PFGE samples. We added an inactivation step for DNase using formaldehyde, and the bacterial strains were embedded in thinner plug molds than usual. These modifications were effective with all strains, and the complete PFGE pattern was obtained in the samples treated with formaldehyde and using thin plug molds. We established a rapid method to obtain a high-quality PFGE result for S. marcescens.