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Use of a heavy inoculum in the in vitro evaluation of the anti-staphylococcal activity of 19 cephalosporins.

The in vitro activity of 19 cephalosporins against 105 clinical isolates of Staphylococcus aureus and S. epidermidis was determined by using a heavy inoculum, i.e., 10(8) to 10(9) organisms per ml, to maximally challenge the antibiotics. The anti-staphylococcal activities of cephaloridine and 87/312 were consistently decreased by the use of a heavy inoculum when compared with the activity obtained with two less-concentrated inocula. The activity of most of the other compounds was also decreased with the use of a heavy inoculum, but this was observed only with selected isolates. Cephapirin, cephalothin, and cefazaflur were the most active drugs against the methicillin-susceptible isolates. Cephaloridine, cefamandole, cefazaflur, and 87/312 had substantial activity against methicillin-resistant staphylococci even with heavy inocula. With the exception of cefaclor against S. aureus, the orally absorbed cephalosporins were generally one-half to one-sixteenth as active as the parenterally administered cephalosporins. The median minimal inhibitory concentrations of five of the 12 parenteral cephalosporins were lower with the methicillin-susceptible S. aureus than with the methicillin-susceptible S. epidermidis strains.

Bacteriological Techniques↗

Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci.

Total RNA was used to study the effect of penicillinase plasmid pI524 and of mecR, the regulatory region located on the methicillin resistance determinant (mec), on the expression of mecA, the gene coding for the low-affinity penicillin-binding protein PBP2', in methicillin-resistant staphylococci. In the present report, we show that the regulation of methicillin resistance occurs primarily at the level of mecA transcription and that in the presence of intact plasmid pI524 or mecR, the gene undergoes negative control. The relative amount of mecA mRNA present during exponential growth in uninduced cultures matches the type of mecA regulation and decreases in the following order: constitutive greater than pI524 greater than mecR-dependent mecA expression. Induction of mecA by methicillin is faster in pI524- than in mecR-controlled strains. The overall mRNA half-life is similar for all strains analyzed. Our results indicate that methicillin resistance under mecR control in certain staphylococcal strains could escape detection by the standard disk diffusion test and broth microdilution test because of the very slow derepression of the mecA gene. This finding is of importance for the clinical detection of this type of methicillin resistance.

Methicillin↗

Pharmacodynamic interaction between RP 59500 and gram-positive bacteria infecting fibrin clots.

The fibrin clot penetration and in vivo bactericidal activity of RP 59500, a new semisynthetic streptogramin, for two Staphylococcus aureus strains (one methicillin resistant and the other methicillin susceptible), two Staphylococcus epidermidis strains (one methicillin resistant and the other methicillin susceptible), and one Enterococcus faecalis strain were evaluated. The clots, inserted subcutaneously, were infected with a mean of 10(8) CFU of the pathogen per g. For each strain, groups of four rabbits received a single intravenous injection of 50 mg of RP 59500 per kg of body weight over 30 min. The mean peak level of RP 59500 in serum in the infected rabbits was 61.9 +/- 6.3 micrograms/ml. The drug was detectable in serum at a level of 0.8 micrograms/ml up to 4 h after administration. The mean peak fibrin clot drug level at 1 h was 3.3 +/- 0.1 micrograms/g. At 6 h, the level in clots was 1.2 +/- 0.1 micrograms/g. The mean half-life in serum in infected rabbits was 0.34 +/- 0.01 h, while in clots the drug exhibited a longer half-life of 3.8 +/- 0.4 h. In vivo, this new streptogramin sterilized the clots infected with the two S. aureus strains studied in less than 1 h and induced a marked reduction in colony counts of the two S. epidermidis strains studied for up to 24 h. The activity of the streptogramin against E. faecalis was limited. These results suggest that RP 59500 should be further evaluated for the treatment of infection with methicillin-resistant staphylococci.

Animals↗

Single-dose pharmacokinetics and antibacterial activity of daptomycin, a new lipopeptide antibiotic, in healthy volunteers.

Three separate single-dose studies were performed to define the disposition and pharmacokinetics of daptomycin in healthy volunteers. Daptomycin was administered as a single 14C-labeled dose (1.0 mg/kg of body weight) and as single doses between 0.5 and 6.0 mg/kg. All doses were intravenous. Antibacterial activity was determined from doses of 2.0, 3.0, 4.0, and 6.0 mg/kg against two strains of Staphylococcus aureus (one methicillin resistant) and one Enterococcus strain. After administration of 14C-labeled daptomycin, recovery of 14C in urine and feces accounted for 83% of the administered dose, with the greatest fraction (78%) appearing in the urine. Specific analysis for daptomycin in both urine and plasma indicated that metabolic products were present in urine, but total 14C in plasma consisted of daptomycin only. Doses between 0.5 and 6 mg/kg were linear, with a limited total body clearance (0.13 to 0.21 ml/min/kg) and a small volume of distribution (0.10 to 0.15 liter/kg). The small volume of distribution may be a factor of the high plasma protein binding (90 to 95%). Renal clearance made up 34 to 54% of total body clearance. Daptomycin demonstrated in vivo antibacterial activity against all three test strains, with the greatest activity observed against methicillin-resistant S. aureus. The predicted MIC for all three strains was approximately 13 micrograms/ml, corresponding to total (bound plus unbound) drug. On the basis of the drug's pharmacokinetics and antibacterial activity, doses of 4 to 6 mg/kg/day, possibly in divided doses, are predicted to be effective.

Adult↗

In vitro activity of OPC-17116.

The in vitro activity of OPC-17116, a new C-5 methyl fluoroquinolone, was compared with the activities of other fluoroquinolones. OPC-17116 inhibited 50% of the members of the family Enterobacteriaceae tested and 90% of Haemophilus influenzae, Neisseria species, and Moraxella catarrhalis isolates at less than or equal to 0.25 microgram/ml. At less than or equal to 2 micrograms/ml, 90% of the Enterobacteriaceae were inhibited, which was comparable to or better than the activities of fleroxacin, ofloxacin, and lomefloxacin but less than the activity of ciprofloxacin. OPC-17116 inhibited 90% of the staphylococci tested at less than or equal to 0.25 micrograms/ml, but it did not inhibit methicillin-resistant, ciprofloxacin-resistant Staphylococcus aureus or Staphylococcus epidermidis. Group A, B, C, F, and G streptococci and Streptococcus pneumoniae were inhibited by less than or equal to 0.5 microgram/ml, being four-fold more active than ciprofloxacin and ofloxacin. Tosufloxacin was the most active agent tested against gram-positive cocci. OPC-17116 inhibited Bacteroides fragilis at 4 micrograms/ml. There was a minimal effect of inoculum size on MIC, and the MBCs were within 1 dilution of the MICs. The activity of OPC-17116 was decreased at pH 6 and in the presence of high Mg2+ concentrations, but it was unaffected by human serum. OPC-17116 showed a postantibiotic effect against Pseudomonas aeruginosa and Staphylococcus aureus similar to the postantibiotic effects reported for other fluoroquinolones. The frequency of spontaneous single-step resistance was low (less than 10(-9)), but repeated passage of organisms in the presence of OPC-17116 resulted in the selection of resistant isolates.

Anti-Infective Agents↗

Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates.

DU-6859, (-)-7-[(7S)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6- fluoro-1-[(1R,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinol one-3- carboxylic acid, is a new fluoroquinolone with antibacterial activity which is significantly better than those of currently available quinolones. The MICs for 90% of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates (MIC90s) were 0.1, 3.13, 0.1, and 0.39 microgram/ml, respectively. MIC50s of DU-6859 against quinolone-resistant, methicillin-resistant S. aureus were 8-, 32-, 64-, and 128-fold lower than those of tosufloxacin and sparfloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DU-6859 inhibited the growth of all strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.1 and 0.2 microgram/ml, respectively, and was more active against enterococci than the other quinolones tested. Although the activity of DU-6859 against Pseudomonas aeruginosa was roughly comparable to that of ciprofloxacin at the MIC50 level, it was fourfold more active than ciprofloxacin at the MIC90 level. DU-6859 was also more active against other glucose-nonfermenting bacteria, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae, than the other drugs tested. Strains of Bacteroides fragilis and Peptostreptococcus spp. were susceptible to DU-6859; MIC90s were 0.39 and 0.2 microgram/ml, respectively. DU-6859 generally showed activities twofold or greater than those of ciprofloxacin and the other drugs against almost all members of the family Enterobacteriaceae. The action of DU-6859 against the clinical isolates was bactericidal at concentrations near the MICs. DU-6859 activity was not affected by different media, pH, inoculum size, or human serum but was decreased in human urine.

Anti-Infective Agents↗

Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus.

Three-factor genetic crosses performed by transformation have shown that the methicillin resistance determinant of Staphylococcus aureus strain DU4916 (the mec-4916 marker) is linked to a novobiocin resistance (Novr) marker (nov-142) and mutational sites affecting pyrimidine (pyr-141), purine (pur-102), and histidine (hisG15) biosynthesis in S. aureus strain 8325. The linkage group thus defined is pyr-141-hisG15-nov-142-pur-102-mec-4916. Phage 80alpha previously propagated on a novobiocin-resistant, methicillin-sensitive (Mecs) 8325 strain was used to infect 21 novobiocin-sensitive, methicillin-resistant clinical isolates (including strain DU4916). Among the novobiocin-resistant transductants so obtained from each recipient, between 1 and 5% were methicillin sensitive (reflecting cotransduction of Novr and Mecs). These results are consistent with the genetic determinant of methicillin resistance having a single chromosomal locus in most, if not all, strains of S. aureus.

Chromosome Mapping↗

Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus.

A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.

Bacteriolysis↗

Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates.

Analysis of DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) was employed to assist in the epidemiologic investigation of the emergence and spread of ciprofloxacin-resistant Staphylococcus aureus at the Atlanta VA Medical Center because many isolates of interest were nontypeable by phages and harbored few plasmids useful as strain markers. Chromosomal DNAs of selected S. aureus isolates were digested initially with 20 different restriction enzymes. EcoRI appeared to give the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Overall, 15 different ribotypes were seen among the 50 S. aureus isolates studied (7 ribotypes among 13 methicillin-susceptible S. aureus [MSSA] isolates and 9 ribotypes among 37 methicillin-resistant S. aureus [MRSA] isolates). Seven of eight ciprofloxacin-resistant MSSA (CR-MSSA) patient isolates had identical antibiograms, were nontypeable by phages, and had a single 22-MDa plasmid. Six of these seven CR-MSSA isolates had an identical ribotype pattern. Ribotyping distinguished this CR-MSSA strain or clone from MRSA and other MSSA isolates, including nontypeable isolates that contained a 22-MDa plasmid. Five ciprofloxacin-susceptible MSSA isolates studied had five ribotypes; one pattern was identical to the CR-MSSA clone. Twenty-three CR-MRSA isolates recovered from the Atlanta VA Medical Center had four different ribotypes. Ribotyping proved to be a useful molecular epidemiologic tool in the study of S. aureus because it differentiated isolates which were indistinguishable by more traditional methods. In addition, this technique demonstrated that at our institution, ciprofloxacin resistance emerged in multiple strains of MRSA, as opposed to primarily a single strain or clone of MSSA.

Bacterial Typing Techniques↗

Genomic and virulence characteristics of Staphylococcus aureus isolates from foodborne outbreak cases.

This study aimed to investigate the genomic characteristics, enterotoxin production, and antimicrobial resistance profiles of Staphylococcus aureus isolates associated with foodborne outbreaks. A total of 19 bacterial isolates were collected from foodborne outbreaks in Guizhou Province, China between 2014 and 2023. Following biochemical identification, all isolates were confirmed as S. aureus. Phylogenetic analysis divided the 19 strains into seven branches. Enterotoxin production was detected using standard microbiological techniques and immunoassays. Antimicrobial susceptibility was evaluated using the broth microdilution method. Whole-genome sequencing and subsequent bioinformatic analyses were conducted to characterize virulence genes, antimicrobial resistance genes, multilocus sequence typing (MLST) genotypes, and phylogenetic relationships among the isolates. This study found that all strains produced classical staphylococcal enterotoxins, with staphylococcal enterotoxin (SEA) showing the highest detection rate (63.16%). Virulence gene profiling revealed widespread presence of hlb, hlgA, nuc, clfB, spa, and set genes. All strains were resistant to penicillin, with high resistance rates for erythromycin and cefoxitin. Multidrug resistance occurred in 11 of the 19 strains, and 22 resistance genes were identified. MLST analysis showed that ST6 and ST59 were the dominant types, with ST59 methicillin-resistant S. aureus (MRSA) strains displaying stronger resistance and more virulence determinants. These findings provide insights into the virulence, resistance, and molecular epidemiology of S. aureus strains involved in foodborne outbreaks, and may provide useful information for future surveillance and risk assessment.

Staphylococcus aureus↗

Teicoplanin associated gene tcaA inactivation increases persister cell formation in Staphylococcus aureus.

Staphylococcus aureus is part of normal human flora and is widely associated with hospital-acquired bacteremia. S. aureus has shown a diverse array of resistance to environmental stresses and antibiotics. Methicillin-resistant S. aureus (MRSA) is on the high priority list of new antibiotics discovery and glycopeptides are considered the last drug of choice against MRSA. S. aureus has developed resistance against glycopeptides and the emergence of vancomycin-intermediate-resistant, vancomycin-resistant, and teicoplanin-resistant strains is globally reported. Teicoplanin-associated genes tcaR-tcaA-tcaB (tcaRAB) is known as the S. aureus glycopeptide resistance operon that is associated with glycopeptide resistance. Here, for the first time, the role of tcaRAB in S. aureus persister cells formation, and ΔtcaA dependent persisters' ability to resuscitate the bacterial population was explored. We recovered a clinical strain of MRSA from a COVID-19 patient which showed a high level of resistance to teicoplanin, vancomycin, and methicillin. Whole genome RNA sequencing revealed that the tcaRAB operon expression was altered followed by high expression of glyS and sgtB. The RNA-seq data revealed a significant decrease in tcaA (p = 0.008) and tcaB (p = 0.04) expression while tcaR was not significantly altered. We knocked down tcaA, tcaB, and tcaR using CRISPR-dCas9 and the results showed that when tcaA was suppressed by dCas9, a significant increase was witnessed in persister cells while tcaB suppression did not induce persistence. The results were further evaluated by creating a tcaA mutant that showed ΔtcaA formed a significant increase in persisters in comparison to the wild type. Based on our findings, we concluded that tcaA is the gene that increases persister cells and glycopeptide resistance and could be a potential therapeutic target in S. aureus.

MRSA↗

Antimicrobial resistance: patterns and trends in the National University Hospital, Singapore (1989-1991).

The commonly isolated organisms, Staphylococcus aureus, Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, Acinetobacter species, Proteus species and Enterobacter species from clinical material other than blood, cerebrospinal fluid and stool, were analysed for their incidence and increasing trends of resistance to the commonly used antimicrobials. Staphylococcus aureus was tested against penicillin, methicillin, erythromycin, gentamicin and co-trimoxazole; Pseudomonas aeruginosa against amikacin, ceftazidime, gentamicin, piperacillin and cefsulodin; and gram negative bacilli against ampicillin, co-trimoxazole, cephalexin, cefuroxime, ceftriaxone, nalidixic acid and nitrofurantoin. Methicillin sensitive Staphylococcus aureus exhibited a high degree of resistance to penicillin only (83%), but methicillin resistant Staphylococcus aureus (34-46%) showed nearly 100% resistance to all drugs tested except for co-trimoxazole over the period of study. A high incidence of resistance was found among Klebsiella species, Enterobacter species, Acinetobacter species and Pseudomonas species. Increasing trends of resistance against cephalosporins were noticed with respect to Acinetobacter species, Klebsiella species and Enterobacter species for cefuroxime and ceftriaxone; Pseudomonas aeruginosa for ceftazidime and cefsulodin. The overall resistance of organisms is notably high. Methicillin-resistant S. aureus is endemic and accounts for about 39% of all S. aureus isolates. The typical nosocomial organisms like Acinetobacter species and Klebsiella species are increasingly developing resistance to useful drugs such as gentamicin, cephalexin and ceftriaxone.

Cephalosporins↗

[Survey of bacterial resistance in Shanghai district].

The data on bacterial resistance monitoring of 5,457 clinical isolates in 1990 against 21 antimicrobial agents showed that most isolates were highly resistant to commonly used antimicrobial agents, such as penicillin, ampicillin, erythromycin, chloramphenicol, tetracycline and gentamicin, with a resistant rate of around 50% or more. Strains resistant to some newer agents, such as cefotaxime, ceftazidime, amikacin and norfloxacin, have appeared and are increasing in number. Multi-resistant strains, i.e. strains resistant to five more antimicrobial agents existed in various bacterial species. Vancomycin was the most active antibiotic against methicillin-resistant staphylococci and no resistant strains were found. Strains of Salmonella and Shigella spp. were still very sensitive to the antimicrobial agents tested, with a sensitive rate of more than 90%. 80% of 765 gentamicin-resistant strains of Enterobacteriaceae were still sensitive to amikacin.

Ampicillin Resistance↗

Problems and dilemmas of antimicrobial resistance.

An important obstacle to the long-term efficacy of an antimicrobial agent is the appearance and spread of resistance to the agent. The fact that many antimicrobials are produced by microorganisms in nature may provide long-term selective pressure for the emergence of resistance in antibiotic-producing as well as -nonproducing organisms. Indeed, the rapidity with which many resistances have appeared after the introduction of a new antibiotic suggests that these resistance genes were already present somewhere in nature prior to clinical use. In the hospital setting, the most recent worrisome resistance traits to emerge include plasmid-mediated resistance to imipenem and to third-generation cephalosporins among nosocomial gram-negative bacteria, and the acquisition of resistance to vancomycin by enterococci. Methicillin-resistant staphylococci continue to be a problem and are increasingly resistant to numerous other agents such as rifampin and the newer fluoroquinolones. The most important resistances seen in community-acquired organisms include beta-lactam resistance in pneumococci and combined ampicillin and chloramphenicol resistance in Haemophilus influenzae. Shigellae resistant to essentially all commonly used oral agents are also a problem, particularly in developing countries. No end is in sight to the problem of antimicrobial resistance, and thus new strategies to prevent infections and control resistant organisms continue to be necessary.

Anti-Bacterial Agents↗

[In vitro kinetics of the activity of fusidic acid and fluoroquinolones combinations against staphylococci according to methicillin-resistance phenotype. Implications for the treatment of osteoarticular infections].

The kinetics of the effect of fusidic acid and of two fluoropiperazinyl quinolones (ofloxacin and pefloxacin), used alone or in combination, on ten methicillin-susceptible or methicillin-resistant strains of staphylococci were studied. Little or no bactericidal effects were seen with fusidic acid. With ofloxacin and pefloxacin, the bactericidal effect was delayed and occurred only with high concentrations. Used in combination, fusidic acid and the fluoroquinolones exhibited no synergistic effects and occasionally antagonized each other in vitro. The methicillin-resistance phenotype of organisms had no influence on results.

Bone Diseases↗

[Considerations on MRSA infections in relation to modern chemotherapy].

Before the antibiotic era of medicine began about 50 years ago, the prognosis for patients with severe staphylococcal infections was extremely poor. In the early 1940s, the introduction of penicillin G (PCG) temporarily solved the problem of staphylococcal infections, but the continued use of this agent caused the selection of resistant strains, which produced penicillinase. Accordingly, the value of PCG had seriously reduced by 1948. By then S. aureus had acquired multiple resistance to virtually all available systemic antibiotics, including aminoglycosides, tetracycline, chloramphenicol and the macrolides. The introduction of the semisynthetic penicillinase resistant penicillins (e.g., methicillin, oxacillin) and the 1st generation cephems brought about a general decline in the prevalence of multiply resistant S. aureus during the early 1960s. The strains of methicillin-resistant S. aureus (MRSA) were initially detected in 1961 in the United Kingdom, shortly after methicillin came into clinical use, and they have subsequently been isolated in many other parts of the world including Japan. In Japan, most strains of MRSA now represent high level resistance to all antibiotics, except for a few antibiotics, such as vancomycin and arbekacin, and therefore, they posed a serious problem clinically and epidemiologically, via immuno-suppressed inpatients, in particular, as the causative pathogen of nosocomial infection. The appearance and spread of such MRSA strains in Japan may be due to the extensive use of broad spectrum antibiotics, particularly the 3th generation cephems. It is now important to reconsider the modern chemotherapy which evoke serious drug-resistant mutants, such as MRSA.

Anti-Bacterial Agents↗