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Pyrithiamine adaptation of Staphylococcus aureus. I. Adaptation and carbohydrate utilization.

Das, S. K. (University of Calcutta, Calcutta, India) and G. C. Chatterjee. Pyrithiamine adaptation of Staphylococcus aureus. I. Adaptation and carbohydrate utilization. J. Bacteriol. 83:1251-1259. 1962.-Staphylococcus aureus has been adapted to pyrithiamine, a thiamine analogue; as a result of this adaptation, the color of the pigment of the organism changes from orange-yellow to lemon-yellow. The adaptation is reversible; the adapted strain will revert after repeated subculture in a medium containing thiamine and no pyrithiamine. Of the major biochemical alterations resulting from adaptation, severe depression in glucose utilization and simultaneous stimulation of acetate utilization have been noticed. The effect of metabolic inhibitors on the utilization of glucose and acetate has also been studied. By measuring the rate of formation of C(14)O(2) from glucose-1-C(14) and glucose-6-C(14), it has been observed that the reduction in C(14)O(2) formation from glucose-1-C(14) by the adapted organism is much more than that obtained from glucose-6-C(14), causing thereby a decreased metabolic ratio of these two substrates after such adaptation. Relative to the normal strain, the adapted strain utilizes acetate-C(14) at a much faster rate, both in the formation of C(14)O(2) and also in the incorporation of C(14) into the protein and lipid fractions; the rate of formation of C(14)O(2) from pyruvate-1-C(14) is not greatly altered. It has been postulated that there is a partial blocking of the pentose phosphate cycle, because of the lowered glucose-1-C(14) utilization, and simultaneous stimulation of the tricarboxylic acid cycle; or perhaps the initiation of some other route after pyrithiamine adaptation would account for the great increase in acetate utilization.

Acclimatization↗

Selective medium for carbohydrate-utilizing transductants of Staphylococcus aureus.

Murphey, W. H. (The University of Texas, Dallas), and E. D. Rosenblum. Selective medium for carbohydrate-utilizing transductants of Staphylococcus aureus. J. Bacteriol. 87:1198-1201. 1964.-The composition and properties of modified eosin-methylene blue agar are described. It is an efficient, selective medium for isolation of carbohydrate-utilizing staphylococci from large, nonutilizing populations. The medium has been used to measure the rate of transduction of mannitol fermentation and to demonstrate genetic recombination in mannitol-negative mutants of Staphylococcus aureus.

Carbohydrate Metabolism↗

Technical aspects of the Staphylococcus aureus teichoic acid antibody assay: gel diffusion and counterimmunoelecrophoretic assays, antigen preparation, antigen selection, concentration effects, and cross-reactions with other organisms.

Because variable results are being reported from laboratories performing the teichoic acid antibody assay in patients with serious infections due to Staphylococcus aureus, we have thoroughly reviewed all technical aspects of the test. This paper reports on the importance of agar and antigen preparation in standardizing results of the assay and reducing the prevalence of false-negative and -positive tests. Once standardized, the counterimmunoelectrophoretic method is as accurate as the gel diffusion method for both initial screening and generating titers; practically, however, unless numerous tests are to be performed, the gel diffusion technique will suffice for most purposes. The cell wall of the Lafferty strain of S. aureus was used as the standard antigen in assays for serum antibodies. We studied whether it is an appropriate antigen and found that antibody titers obtained with the Lafferty strain antigen were, in three patients with endocarditis, the same as those obtained with antigens from the individual blood stream isolates. We have also confirmed that pooled human gamma globulin can be used, by back titration against newly prepared lots of antigen, to select optimal antigen concentration and is as good as more specific, higher titer serum specimens for that purpose. Finally, cell wall antigens from Staphylococcus epidermidis and a variety of streptococci may react with normal human sera, but such antigens are distinct by immunoprecipitation from those from S. aureus.

Antibodies, Bacterial↗

Immunoglobulin E anti-Staphylococcus aureus antibodies in atopic patients.

Sera from 56 patient and normal adults were examined to quantitate total immunoglobulin E (IgE) and IgE antibodies to Staphylococcus aureus and Staphylococcus epidermidis. Patients were divided into six groups based on clinical symptoms; a seventh group consisted of normal adults. Anti-S, aureus IgE binding was significantly higher in three groups of patients (those with eczema, those with or without series staphylococcal abscesses, and allergic patients with staphylococcal skin infections) than it was in the control group. Patients with high IgE due to allergies or parasitic infections without staphylococcal infections and patients with low or normal IgE and serious staphylococcal infection showed low levels of binding. The assay measured specific binding of IgE to bacterial antigens.

Adult↗

Comparison of rapid identification assays for Staphylococcus aureus.

A total of 137 strains of Staphylococcus species were blindly tested by four rapid serological assays, and the results were compared with those of the tube coagulase assay. For the S. aureus isolates, the Sero-STAT Staph assay gave six false-negative results, four of which were for methicillin-resistant strains. The Accu -Staph, Staphylatex , and Staphyloslide assays identified all the coagulase-positive strains as Staphylococcus aureus. Among the coagulase-negative staphylococci, false-positive results were seen with strains of S. capitis. S. saprophyticus, and S. cohnii. The overall accuracy of the kits compared with the tube coagulase test ranged from 95.1 to 100%.

Bacteriological Techniques↗

Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses.

A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable.

Animals↗

Species-specific and ubiquitous DNA-based assays for rapid identification of Staphylococcus epidermidis.

Staphylococcus epidermidis is an aerobic gram-positive coccus that is now recognized among the coagulase-negative staphylococci as an etiological agent with an important range of pathogenicity in humans. Several diagnostic kits based on biochemical or immunological reactions can efficiently identify Staphylococcus aureus. However, these tests are often unreliable for the identification of coagulase-negative staphylococcal species including S. epidermidis. Since DNA-based assays for the species-specific identification of S. epidermidis remain unavailable, we have developed such tests in order to improve the accuracy and the rapidity of tests for the diagnosis of S. epidermidis infections. On the basis of the results of hybridization assays with clones randomly selected from an S. epidermidis genomic library, we identified a chromosomal DNA fragment which is specific and 100% ubiquitous for the identification of S. epidermidis. This 705-bp fragment was sequenced and used to design PCR amplification primers. PCR assays with the selected primers were also highly specific and ubiquitous for the identification from bacterial cultures of clinical isolates of S. epidermidis from a variety of anatomic sites. While three strains of S. capitis were misidentified as S. epidermidis with the API Staph-Ident system and 2.5% of the S. epidermidis identifications were inconclusive with the MicroScan Autoscan-4 system, the PCR assay was highly specific and allowed for the correct identification of all 79 S. epidermidis strains tested. The PCR assays developed are simple and can be performed in about 1 h. The DNA-based tests provide novel diagnostic tools for improving the diagnosis of S. epidermidis infections.

Bacteriological Techniques↗

Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay.

A rapid procedure was developed for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g., nose or inguinal swabs). After a rapid conditioning of samples, the method consists of two main steps: (i) immunomagnetic enrichment in S. aureus and (ii) amplification-detection profile on DNA extracts using multiplex quantitative PCR (5'-exonuclease qPCR, TaqMan). The triplex qPCR assay measures simultaneously the following targets: (i) mecA gene, conferring methicillin resistance, common to both S. aureus and Staphylococcus epidermidis; (ii) femA gene from S. aureus; and (iii) femA gene from S. epidermidis. This quantitative approach allows discrimination of the origin of the measured mecA signal. qPCR data were calibrated using two reference strains (MRSA and methicillin-resistant S. epidermidis) processed in parallel to clinical samples. This 96-well format assay allowed analysis of 30 swab samples per run and detection of the presence of MRSA with exquisite sensitivity compared to optimal culture-based techniques. The complete protocol may provide results in less than 6 h (while standard procedure needs 2 to 3 days), thus allowing prompt and cost-effective implementation of contact precautions.

Bacterial Proteins↗

Infection of a ventriculoatrial shunt with phenotypically variable Staphylococcus epidermidis masquerading as polymicrobial bacteremia due to various coagulase-negative Staphylococci and Kocuria varians.

The diagnosis of bloodstream infection with coagulase-negative staphylococci is frequently based on the isolation of the same organism from more than one blood culture. Phenotypic variation is a common characteristic of pathogenic strains of Staphylococcus epidermidis which may affect species identification by the microbiology laboratory. We describe a patient with a new onset of nephritis and gram-positive bacteremia. Gram-positive cocci grew in multiple blood cultures and were identified by the Vitek 2 system as Kocuria varians, Staphylococcus hyicus, and S. epidermidis. Bacterial isolates grew on blood agar and Congo red agar plates as two distinct morphotypes and exhibited phenotypic variation. Neither morphotype could be identified by the API-Staph assay. Cellular fatty acid analysis identified one of the morphotypes as S. epidermidis but could not identify the other morphotype. All isolates were found to be identical by pulsed-field gel electrophoresis, and both colonial morphotypes were identified as S. epidermidis by 16S ribosomal gene sequencing. Phenotypic variation of S. epidermidis may affect identification to the species level by phenotype-based identification systems. Caution should be exercised when differentiating between true infection and contamination based on strain identification.

Actinomycetales↗

Evaluation of the LightCycler Staphylococcus M GRADE kits on positive blood cultures that contained gram-positive cocci in clusters.

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.

Bacterial Typing Techniques↗

The inoculum effect of methicillin-susceptible Staphylococcus aureus on cefazolin and other antimicrobial agents.

UNLABELLED: The inoculum effect (IE) refers to a reduced susceptibility of methicillin-susceptible Staphylococcus aureus (MSSA) to certain antibiotics under high bacterial inocula and may contribute to treatment failure. This exploratory study assessed IE prevalence among 234 nonduplicate MSSA isolates across 11 agents spanning major therapeutic classes, including cefazolin, and characterized IE-positive clones via whole-genome sequencing to inform clinical strategies. Minimum inhibitory concentrations (MICs) were determined by broth microdilution at standard and high inocula. Whole-genome sequencing was performed on IE-positive strains to identify β-lactamase types and conduct multilocus sequence typing. The highest prevalence of IE was observed for trimethoprim-sulfamethoxazole (9.4%), followed by erythromycin (8.8%), linezolid (6.8%), penicillin (6.1%), clindamycin (5.5%), vancomycin (3.8%), cefazolin (3.0%), levofloxacin (1.5%), tetracycline (0.5%), and oxacillin and gentamicin (0.0%). All cefazolin IE-positive strains carried blaZ type A, and ST25 was the most common sequence type (42.9%). For trimethoprim-sulfamethoxazole, erythromycin, and clindamycin IE, ST7 was the most common sequence type (22.7%, 26.7%, and 33.3%, respectively). ST1281 and ST188 were the predominant sequence types among strains exhibiting linezolid IE and vancomycin IE (25.0% and 33.3%, respectively). Among the 234 MSSA strains, 66.7% of ST59, 60.0% of ST25, 58.3% of ST5, and 54.2% of ST7 strains exhibited IE to at least one antimicrobial agent. Cefazolin IE was associated with blaZ type A, and ST5, ST7, ST59, and ST25 were the major sequence types associated with IE across the antimicrobial classes tested. IMPORTANCE: Methicillin-susceptible Staphylococcus aureus (MSSA) can show an inoculum effect on multiple antimicrobial agents, which may reduce antibiotic activity under high-burden conditions. In this study, MSSA isolates from Shanghai exhibited inoculum effects on several commonly used agents, although the overall detection rates were low. Cefazolin inoculum effect was specifically associated with blaZ type A, and several major sequence types were more likely to exhibit this phenotype. These findings improve our understanding of the epidemiology of the inoculum effect in MSSA and may help guide laboratory detection and antimicrobial treatment decisions.

Cefazolin↗

A comparative analysis of the clinical and genomic characteristics of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in Korea and Japan.

Panton-Valentine leukocidin (PVL) is a leukocyte-lytic toxin produced by Staphylococcus aureus, which is primarily associated with skin and soft tissue infections. Notably, there has been an increase in the number of cases caused by the USA300 lineage in Japan. However, the reported prevalence of USA300 is limited in other Asian countries, including Korea. This study investigated the prevalence of PVL-positive methicillin-resistant S. aureus (MRSA) in Korea and Japan and compared their molecular epidemiological characteristics. A total of 463 MRSA strains were analyzed, comprising 283 strains from patients visiting two hospitals in Seongnam and Seoul, Korea, and 180 strains from six hospitals in Tokyo, Japan, between 2018 and 2019. The PVL-encoding genes lukS/F-PV were detected using PCR. Molecular epidemiological and phylogenetic analyses were performed using next-generation sequencing. Overall, 27 (9.5%) PVL-positive MRSA strains were detected among strains isolated from Korea, and 16 (8.9%) were detected among those isolated from Japan. Genotyping of PVL-positive strains revealed that 85.2% of Korean and 81.3% of Japanese strains belonged to USA300. Most USA300 strains from Japan and Korea formed distinct clusters in phylogenetic analyses. Meanwhile, ΨUSA300 and ST22-PT, clones that are prevalent in Japan, were isolated in Korea. This study showed that USA300 strains, which are becoming more prevalent in Japan, are also present in Korea. Furthermore, this study suggests that ΨUSA300 and ST22-PT may be spreading between these two countries. Therefore, it is necessary to continue monitoring the epidemiological trends of PVL-positive MRSA clones both domestically and internationally.IMPORTANCEPanton-Valentine leukocidin (PVL) is a major toxin produced by Staphylococcus aureus. Although a rapid increase in PVL-positive strains has been reported in Japan, data on PVL-positive strains in Korea remain limited. In this study, we performed a comparative analysis of PVL-positive S. aureus isolates from Korea and Japan. The results showed that the clinical backgrounds and genetic profiles of PVL-positive strains isolated in Korea and Japan were highly similar. Furthermore, we confirmed for the first time that clones circulating in Japan, including ΨUSA300 and ST22-PT, were also isolated in Korea. These findings provide valuable insights into the epidemiological status of PVL-positive S. aureus in East Asia.

Leukocidins↗

Mocravimod as a repurposing drug against clinical isolates of Staphylococcus aureus by targeting cell membrane.

UNLABELLED: Staphylococcus aureus infections, particularly those caused by multidrug-resistant strains and associated with biofilm formation, pose a major therapeutic challenge in clinical practice. The objective of this study was to evaluate the antibacterial and antibiofilm activity of mocravimod (KRP-203), an FDA-approved S1P receptor modulator, against clinical S. aureus isolates and to explore its underlying mechanism of action. The antibacterial activity of KRP-203 was assessed against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) using MIC determination, time-kill assays, and biofilm inhibition models. KRP-203 exhibited strong bactericidal activity against planktonic MSSA and MRSA, with MIC values ranging 6.25-50μM. Time-kill assays demonstrated rapid bacterial eradication at 8× MIC within 2 h, showing superior killing kinetics compared with vancomycin. At sub-inhibitory concentrations, KRP-203 inhibited biofilm formation by up to 70% and reduced viable bacterial counts in mature biofilms by >2.5 logs. To elucidate the antibacterial mechanism, whole-genome sequencing and quantitative proteomic analyses were performed. These analyses revealed mutations in membrane-associated genes, including glnQ and BCAT, and significant alterations in proteins related to membrane integrity and redox regulation. Consistently, functional assays confirmed that KRP-203 disrupts bacterial cell membrane, as evidenced by dose-dependent membrane depolarization, increased permeability, and direct binding to cardiolipin and phosphatidylglycerol. Molecular docking further predicted a favorable interaction between KRP-203 and GlnQ. In conclusion, KRP-203 demonstrated notable antibacterial and antibiofilm activity against S. aureus, likely through membrane integrity disruption. While these findings highlight its potential as a repurposed antibacterial agent, further studies are required to fully elucidate its molecular targets, optimize antibacterial efficacy, and evaluate its in vivo safety profile. IMPORTANCE: Antibiotic resistance and the formation of biofilms, which protect bacteria from medications and immunological responses, present the significant challenges for the clinical treatment of Staphylococcus aureus infections. This study reveals mocravimod hydrochloride (KRP-203), a clinically approved drug initially intended to treat leukemia, as a viable new candidate against S. aureus infection. KRP-203 quickly kills both drug-susceptible and resistant S. aureus, including difficult-to-treat biofilm-associated cells. Its membrane-disrupting activity quickly kills drug-resistant bacteria while also destroying biofilm formations, presenting a dual action rarely accomplished by conventional antibiotics. Critically, KRP-203's established safety profile in human studies may hasten its repurposing as a new weapon against biofilm-associated infections, providing possible solutions for chronic and drug-resistant S. aureus infections where existing treatments commonly fail.

Biofilms↗

Coagulase-negative strains of staphylococcus possessing antigen 51 as agents of urinary infection.

A group of strains of Staphylococcus albus is described which produced neither coagulase nor haemolysin, was slightly sensitive or resistant to novobiocin, and sensitive to all other antibiotics, to sulphonamides, and to nitrofurantoin. The agglutinating antigen 51 was isolated from all strains from patients with urinary infections and abundant pyuria. In more than 40 cases studied it was not possible to isolate any other bacterial agent, and the cure of clinical symptoms always coincided with the disappearance of the coagulase-negative staphylococcus strain.

Anti-Bacterial Agents↗

Bacteraemia due to recurrent reinfection with Staphylococcus epidermidis associated with defective opsonisation and procidin function in serum.

AIMS: To differentiate between reinfection and relapsing infection with Staphylococcus epidermidis in a middle-aged woman with defective opsonisation and procidin function in serum. METHODS: Microbiological typing was done by biotyping, phage typing, and polyacrylamide gel electrophoresis of radiolabelled bacterial proteins (radioPAGE method). Polymorphonuclear cell function was assessed in vitro by phagocytosis and killing of Candida albicans; measurement of neutrophil random locomotion and chemotaxis; reduction of nitroblue tetrazolium after stimulation by opsonised Candida and a radiometric saccharomyces opsonisation assay. The effect of plasma infusions on opsonic activity was assessed by chemiluminescence using control polymorphonuclear leucocytes with a laboratory strain of S epidermidis opsonised with either patient or control serum. RESULTS: Recurrent reinfection with different strains of Staphylococcus epidermidis rather than relapsing infection was confirmed as having occurred by typing bacterial strains. The RadioPAGE method detected all the S epidermidis strains involved in this patient's illness. The patient's serum was shown to be defective in both opsonin and procidin function. The defects were correctable in vitro by the addition of normal serum. Clinical recovery occurred after repeated infusions of normal fresh frozen plasma and prolonged antibacterial treatment; antibacterial treatment alone was insufficient. CONCLUSIONS: The radioPAGE method is useful in distinguishing recurrent reinfection with S epidermidis from relapsing infection with this organism. Elucidation of the nature of, and underlying predisposition to, infection in the patient studied allowed a rational treatment plan of plasma infusion combined with antibacterial treatment to be devised which ultimately proved successful.

Autoradiography↗

In vitro activity effects of combinations of cephalothin, dicloxacillin, imipenem, vancomycin and amikacin against methicillin-resistant Staphylococcus spp. strains.

BACKGROUND: combinations of drugs has been proposed as an alternative for oxacillin-resistant staphylococci infections, however, limited information about in vitro combinations are available for multi-resistant strains. The objective of this study was to describe the interaction of beta-lactams in combination with vancomycin or amikacin against 26 oxacillin and amikacin-resistant nosocomial Staphylococcus spp. isolates. METHODS: activity of dicloxacillin plus amikacin, cephalothin plus amikacin, cephalothin plus vancomycin, imipenem plus vancomycin and vancomycin plus amikacin was evaluated by checkerboard synergy tests and the fractional inhibitory concentration index (FIC) was calculated. RESULTS: dicloxacillin plus amikacin, and cephalothin plus amikacin were synergistic or partially synergistic in 84.6% and 100% respectively. For nearly half of the isolates the mean concentrations of dicloxacillin, cephalothin and amikacin at which FIC indexes were calculated were achievable therapeutically. Vancomycin plus amikacin had synergistic effect only against two isolates, and partially synergistic in 38.6%. For the combinations vancomycin plus cephalothin and vancomycin plus imipenem the effect was additive in 76.9% and 80.7% respectively. CONCLUSION: in this study the checkerboard analysis showed that amikacin in combination with cephalothin or dicloxacillin was synergistic against most of the resistant strains of S. aureus and coagulase-negative Staphylococcus. Vancomycin in combination with a beta-lactam (cephalothin or imipenem) showed additivity. An indifferent effect predominated for the combination vancomycin plus amikacin. Even though a synergistic effect is expected when using a beta-lactam plus amikacin combination, it is possible that the effect cannot be clinically achievable. Careful selection of antimicrobial combinations and initial MICs are mandatory for future evaluations.

Amikacin↗

Persistent bacteremia and severe thrombocytopenia caused by coagulase-negative Staphylococcus in a neonatal intensive care unit.

OBJECTIVE: Coagulase-negative Staphylococcus (CoNS) is the most frequent cause of late-onset sepsis in NICUs, but mortality is rare and morbidity is unusual. We report a new syndrome of CoNS sepsis characterized by significant morbidity and persistent bacteremia despite aggressive antibiotic therapy and no identified focus of infection. METHODS: We conducted a retrospective review of infants in the NICU with CoNS bacteremia between 2000 and 2002. Statistical analysis included an initial exploratory analysis followed by logistic regression. Microbiological identification of all isolates and molecular typing were performed. RESULTS: Thirty-one neonates with persistent CoNS bacteremia were compared with 60 randomly selected neonates from a group of 140 with nonpersistent CoNS bacteremia. The clinical manifestations at presentation, gestational ages, and birth weights were similar in the 2 groups. Thrombocytopenia was present in 26 (84%) neonates with persistent CoNS bacteremia but only in 8 (13%) neonates in the nonpersistent group. Central venous catheterization increased the risk for persistent CoNS bacteremia, but 42% of the persistent group was never catheterized. Staphylococcus epidermidis was the most common isolate in both groups. Molecular typing failed to identify a predominant clone. CONCLUSIONS: The syndrome of persistent CoNS septicemia is remarkable for thrombocytopenia and persistence in the absence of central venous catheterization. Clinical manifestations at presentation and demographic characteristics did not discriminate between the persistent and nonpersistent groups. We did not identify the emergence of a particularly virulent clone, but it is possible that some strains of CoNS have acquired the capacity to persist under different conditions.

Bacteremia↗

Epidemiology of drug resistance: the case of Staphylococcus aureus and coagulase-negative staphylococci infections.

OBJECTIVE: To study the activity of several antibiotics against Staphylococcus spp. MATERIAL AND METHODS: The study included 1209 strains of Staphylococcus spp. from two institutions; Instituto Nacional de Pediatría (National Institute of Pediatrics) and Hospital Infantil de México Federico Gómez (Mexico City Children's Hospital). Minimum Inhibitory Concentrations of all antibiotics were determined by the agar macrodilution technique and standard methods from the National Committee for Clinical Laboratory Standards. RESULTS: Resistance of S. aureus was 14.2% and that of coagulase-negative staphylococci was 53.4%. The activity of different antibiotics is presented in detail. CONCLUSIONS: Surveillance of strains resistant to methicillin is necessary. The English version of this paper is available too at: http://www.insp.mx/salud/index.html.

Coagulase↗