Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus

Staphylococcus aureus small colony variants (SCVs) occur frequently after local treatment with aminoglycosides and cause persistent as well as recurrent infections. So far, the molecular mechanism of the emergence of SCVs is not understood and regulatory as well as genetic mechanisms seem conceivable. To screen for possible mutations, the hemin biosynthetic gene cluster of a gentamicin-induced SCV was sequenced and was found to contain a deletion in the gene hemH. To further assess the influence of a high mutation rate on the development of SCVs, we tested the emergence of SCVs in a strain that had been inactivated in the DNA proofreading enzyme MutS. In the mutant, spontaneous SCVs emerged 556-fold more frequently than in the parent strain. By incubation in the presence of subinhibitory concentrations of gentamicin, the SCV frequency in the parent strain could be increased to 9.7 x 10(-6), whereas it remained rather stable in the mutant (1.8 x 10(-5)). Eighty percent of the gentamicin-induced SCVs were hemin auxotrophic in contrast to only 20% of the spontaneous SCVs which may explain the large proportion of hemin auxotrophs among clinical SCVs from patients previously treated with aminoglycosides. Additionally, a clinical S. aureus SCV isolate with a mutator phenotype, indicated by the generation of rifampicin-resistant mutants at a 16-fold higher frequency than in the reference strain S. aureus NCTC 8325, was characterized. The results demonstrate that a high mutation rate favours the emergence of SCVs, and suggest that mutations in general play an important role in the development of SCVs.     

Small-colony variants of Staphylococcus aureus

Staphylococcus aureus remains a versatile and dangerous pathogen. Small-colony variants (SCVs) of S. aureus are a naturally occurring sub-population, first described nearly 100 years ago. These variants, of which there are different classes, grow slowly and have many atypical characteristics thought to be due to defective electron transport, and include minute colony forms. SCVs have been isolated from sites of infection, particularly persistent recurrent ones (e.g. in chronic osteomyelitis and cystic fibrosis), and also may arise following exposure to certain antibiotics. During infection the intraendothelial-cell milieu stimulates the formation of SCVs, which can better survive the assault of cell-mediated immunity. SCV phenotypes produce less tissue damage than normal staphylococci. Although microscopic morphology and Gram's staining of SCVs are normal, clinical microbiology laboratories may fail to detect them because of their very slow growth. The full extent of the role of S. aureus SCVs in clinical disease, and the most appropriate means of identifying such strains or testing to predict the clinical usefulness of therapeutic regimens, remains unknown. Failure to recover SCVs results in a major susceptibility reporting error, as the more resistant component of the infection will not have been reported. No controlled trials of therapy have been conducted, and, thus, optimal therapy has yet to be defined. However, SCVs are resistant to aminoglycoside antibiotics, and may be resistant to trimethoprim-sulphmethoxazole. The effectiveness of cell-wall-active antibiotics is reduced. SCVs are transmissible, and current infection control recommendations for normal S. aureus infections are appropriate.     

Crossbreeding channel catfish for improvement of body weight in earthen ponds

Twelve experiments on intraspecific crossbreeding of channel catfish, Ictalurus punctatus, for improvement of body weight are compared and reviewed. Crossbred fingerlings resulting from matings of unrelated F1 crossbred populations did not show heterotic growth. Crossbreds resulting from pure strain P1 showed an average increase of 10.3 percent in growth above the fastest growing parent strain. Marion female X Kansas male, a crossbred from two rapidly growing domestic strains, was the fastest growing to fingerling size. Average increase in body weight (fingerling to harvest size) by crossbreds was 1.5 percent greater than the fastest growing parent strain. Marion X Kansas, Auburn X Kansas, and Auburn X Uvalde were the fastest growing crossbreds to harvest size (8-13 percent increase in growth rate). Six of nine crossbreds made from Pi generations expressed heterosis above both parent strains for body weight. Eight of nine crossbreds grew better than at least one of their parents. Reciprocal crossbreds did not grow at the same rate. Males and females of a specific strain had different combining abilities with other strains. There was a maternal effect for combining ability. All crossbreds made with Auburn females exhibited heterosis.     

Fourier-transform infrared spectroscopic analysis is a powerful tool for studying the dynamic changes in Staphylococcus aureus small-colony variants

Infections due to small-colony variants (SCVs) of Staphylococcus aureus in patients with chronic and recurrent infections are an emerging problem; however, studies with this subpopulation are hampered by the fact that SCVs may exhibit unstable phenotypes, making them difficult to study, particularly in broth media. In this study, two S. aureus sets comprising the (i) normal and the (ii) SCV phenotype (clonal with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding site-directed mutants displaying the SCV phenotype (knockout of hemB) and (iv) their complemented mutants were examined by Fourier-transform infrared (FTIR) spectroscopy. Phenotypes were defined on solid and in broth media. Using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information resulted in a dendrogram with clear discrimination between SCV and normal phenotypes. The SCVs gave an FTIR fingerprint that was easily recognizable and that was much closer to other SCVs than to their parent strains. This technique offers for the first time a noninvasive approach to investigate dynamic processes of reversion of SCVs to the normal phenotype and vice versa. Thus, FTIR spectroscopy allowed a rapid and reproducible tool for the examination of different subpopulations of S. aureus on solid and in broth media for diagnostic and research purposes.     

Effectiveness of hydroxyapatite-vancomycin bone cement in the treatment of Staphylococcus aureus induced chronic osteomyelitis

In the field of local application of antimicrobials, a number of novel drugs and/or new drug delivery systems have been developed in recent years. The present study aimed to investigate hydroxyapatite cement (HAC) as a carrier for vancomycin in the treatment of chronic osteomyelitis due to Staphylococcus aureus strains with various mechanisms of resistance. The release of vancomycin from standard test cylinders was determined in vitro and the efficacy of the delivery system was measured in vivo using a rabbit model of chronic osteomyelitis. First, powdered HAC was mixed with vancomycin at 80, 160 and 240 mg/g. After hardening, formed cylinders were eluted in phosphate buffer and antibiotic release was measured by agar diffusion. High levels of release (1512+/-318 to 1937+/-336 microg/ml) were obtained for 12 to 20 days depending on the dosage of vancomycin. Additionally, bone infection was induced in the tibia of 30 New Zealand white rabbits by injecting either a methicillin-resistant S. aureus strain (MRSA) or a S. aureus strain with a small colony variant (SCV) phenotype. After 3 weeks (chronic infection), all animals were treated by debridement. Moreover, group 1 (challenged with SCVs) and group 2 (challenged with MRSA) were treated by filling the marrow with HAC alone, whereas in groups 3 (SCVs) and 4 (MRSA) the marrow was filled with HAC/vancomycin (160 mg/g). After 6 weeks all animals were sacrificed. At 3 weeks, pathogens were detected in 24 of 30 animals. All swabs of the control groups, positive for S. aureus on day 21, were also positive on day 42 and S. aureus strains recovered were shown to be clonal to the strains used for induction of osteomyelitis. By contrast, no growth was found in the treatment group following 7 days of incubation in BHI bouillon. HAC/vancomycin-treated animals showed no histological evidence of infection on day 42. In the other groups, different stages of chronic osteomyelitis were found histologically. No local or systemic side effects due to HAC or vancomycin were seen. HAC is an effective carrier material for antibiotic compounds even in refractory infections due to MRSA or S. aureus SCVs.     

异质性万古霉素耐药葡萄球菌分离及生物学特性观察

目的　了解本地区临床标本中异质性万古霉素耐药葡萄球菌 (h VRS)分离率 ,并对其生物学特性进行观察。方法　采用琼脂筛选和菌谱分析法对 5 6株甲氧西林耐药的葡萄球菌进行检测 ,同时对分离细菌的生长特性和超微结构进行观察 ,并与同源性敏感菌株及金黄色葡萄球菌标准菌株相比较。结果　本地区h VRS检出率为 14 .3% ,其中血浆凝固酶阴性葡萄球菌的分离率 (2 3.1% )明显高于金黄色葡萄球菌 (6 .7% ) ;耐药亚群与同源性敏感菌株和标准菌株比较 ,生长速度减慢 ,在固体培养基上菌落大小不等 ,在液体培养基中呈沉淀生长 ,电镜观察可见细胞壁增厚。结论　h VRS在本地区的临床标本中有一定的分离率 ,应引起重视 ;该菌的很多生物学特性与同源性敏感菌株有所不同 ,其中细胞壁增厚是比较明显的改变 ,并与该菌的耐药性有关。     

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.     

Small-colony variants (SCVs) of staphylococci: a role in foreign body-associated infections

Staphylococci have various strategies for resisting therapy that extend beyond classic mechanisms. Clinical experience with device-associated infections as well as with infections due to small-colony variants (SCVs) clearly shows that both antibacterial chemotherapy and host defense mechanisms are often unable to eliminate the pathogens and cure these infections. Of particular interest is the fact that in the past few years an increasing number of various foreign body-related infections due to staphylococcal SCVs have been reported. In this overview, the characteristics of SCVs recovered from clinical specimens and of defined mutants displaying the SCV phenotype are described. Their slow growth and changing biochemical and physiological features represent a challenge to clinical laboratory personnel, because recovery, identification, as well as susceptibility testing of these variants need particular efforts. In addition, the reduced susceptibility to aminoglycosides and the ability of SCVs to persist intracellularly require specific attention for the treatment of these infections. Thus, special efforts to search for these variants formed by Staphylococcus aureus or by coagulase-negative staphylococci should be considered when an infection is particularly resistant to therapy, persists for a long period or fails to respond to apparently adequate therapy with antimicrobial compounds.     

Staphylocidal action of thrombin-induced platelet microbicidal protein is not solely dependent on transmembrane potential.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic, antimicrobial peptide released from rabbit platelets when stimulated with thrombin. We studied the relationship between staphylococcal transmembrane potential (delta psi) and tPMP staphylocidal activity. A genetically related pair of Staphylococcus aureus strains, 6850 and JB1, which differ in delta psi generation (-143 and -97 mV, respectively) were used. Mutant JB-1 was substantially less susceptible to tPMP than the parental strain, 6850. Menadione supplementation, which normalized the delta psi of strain JB-1, did not restore JB-1 tPMP susceptibility. These findings suggest that the staphylocidal activities of tPMP require factors other than or in addition to an intact delta psi.     

Infection of polarized airway epithelial cells by normal and small-colony variant strains of Staphylococcus aureus is increased in cells with abnormal cystic fibrosis transmembrane conductance regulator function and is influenced by NF-κB

The infection of nonphagocytic host cells by Staphylococcus aureus and more particularly by small-colony variants (SCVs) may contribute to the persistence of this pathogen in the lungs of cystic fibrosis (CF) patients. The development of chronic infections is also thought to be facilitated by the proinflammatory status of CF airways induced by an activation of NF-κB. The aim of this study was to compare the infection of non-CF and CF-like airway epithelial cells by S. aureus strains (normal and SCVs) and to determine the impact of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and NF-κB on the infection level of these cells by S. aureus. We developed an S. aureus infection model using polarized airway epithelial cells grown at the air-liquid interface and expressing short hairpin RNAs directed against CFTR to mimic the CF condition. A pair of genetically related CF coisolates with the normal and SCV phenotypes was characterized and used. Infection of both cell lines (non-CF and CF-like) was more productive with the SCV strain than with its normal counterpart. However, both normal and SCV strains infected more CF-like than non-CF cells. Accordingly, inhibition of CFTR function by CFTRinh-172 increased the S. aureus infection level. Experimental activation of NF-κB also increased the level of infection of polarized pulmonary epithelial cells by S. aureus, an event that could be associated with that observed when CFTR function is inhibited or impaired. This study supports the hypothesis that the proinflammatory status of CF tissues facilitates the infection of pulmonary epithelial cells by S. aureus.     

Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants

To identify the most rapid and reliable technique for recovery and identification of Staphylococcus aureus small-colony variants (SCVs), the colonial appearance of 106 isolates representing SCVs and the normal phenotype were evaluated on two newly described chromogenic agar media. Although almost all of the SCVs grew on the chromogenic agar media, they did not exhibit a change of color. In comparison with conventional media, S. aureus ID agar (SAID; bioMerieux, La Balme Les Grottes, France) showed the most reliable results, with 49 of 53 SCVs tested growing either as an SCV colony or with a normal phenotype after only 24 h of incubation. Growth of SCVs was often not detected before 72 h of incubation on some of the media tested. In conclusion, the most accurate and rapid method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens onto both Columbia blood agar and SAID.     

Prevalence and genetic diversity of Staphylococcus aureus small-colony variants in cystic fibrosis patients

Staphylococcus aureus small-colony variants (SCVs) are being isolated more frequently in cystic fibrosis (CF) patients. We aimed to determine the prevalence of S. aureus SCVs and their phenotypic and genotypic properties in CF patients admitted to a university hospital. Specimens of 248 patients were examined during a period of 11 months. Colonies supposed to be SCVs were evaluated on Columbia blood agar, mannitol salt agar, and brain–heart infusion agar with 5% NaCl (BHIA 5% NaCl). Strains were confirmed by S. aureus nucA PCR. Antibiotic susceptibilities of SCVs and simultaneously isolated S. aureus strains were determined for oxacillin, gentamicin, trimethoprim–sulphamethoxazole, vancomycin, ciprofloxacin, linezolid, and tigecycline. Genetic relatedness between SCVs and normal S. aureus strains was determined with a pulsed-field gel electrophoresis (PFGE) method. S. aureus SCVs were detected in 20 of 248 patients (8.1%). The highest SCV isolation rate was obtained with MSA, followed by BHIA 5% NaCl. Auxotrophism for thymidine was demonstrated in six SCVs. The tigecycline susceptibilities of 48 SCV strains isolated in this study showed higher MIC values than those of 33 simultaneously isolated normal S. aureus strains. Whereas SCVs and normal S. aureus strains showed identical genotypes in 14 of the patients, five patients showed different genotypes. This first study from Turkey evaluating S. aureus SCVs in CF patients has indicated the importance of considering and reporting SCVs in chronic infections such as CF. The presence of SCVs will probably indicate persistent infection, and this might impact on antibiotic treatment decisions, as they are more resistant to antibiotics.     

Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis

Persistence of Staphylococcus aureus during invasive infections has been associated with a small-colony variant (SCV) phenotype. SCVs are frequently auxotrophic for menadione or hemin, two compounds involved in the biosynthesis of the electron transport chain. SCVs have been shown to be more resistant to antibiotics such as aminoglycosides, grow slowly and persist intracellularly. The aim of this study was to assess the virulence of an hemB mutant, which has been shown to display the typical characteristics of clinical SCVs, in a murine model of septic arthritis. NMRI mice were inoculated intravenously with either the wild type strain Newman or with its mutant displaying the SCV phenotype. The clinical, bacteriological, and histopathological progression of the disease was studied. Mice inoculated with the hemB mutant displayed a higher frequency and a significantly higher severity of arthritis than mice inoculated with the wild type Newman strain. Despite that, the mutant inoculated mice displayed significantly lower bacterial burden in their kidneys and joints compared with mice exposed to the wild parental strain. Notably, the hemB mutant produced almost 20 times more protease in vitro than the parental strain. We conclude that the small colony variants of S. aureus are more virulent on a per organism basis than its isogenic parental strain in the model of septic arthritis. This can at least in part be explained by the ability of SCV to produce high amounts of destructive proteases.     

Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus

Thymidine-dependent small-colony variants (SCVs) of Staphylococcus aureus are frequently associated with persistent and recurrent infections in cystic fibrosis patients. The phenotypic appearance of S. aureus SCVs or normal-colony variants (NCVs) is postulated to be affected by the intracellular amount of dTMP. This hypothesis was proven by metabolic pathway assays revealing altered intracellular dTMP concentrations, followed by investigation of the associated phenotype. Inhibition of the staphylococcal thymidylate synthase, which generated intracellular dTMP from dUMP, using 5-fluorouracil and co-trimoxazole resulted in an SCV phenotype. Inhibition of a nucleoside transporter, which provided the bacterial cell with extracellular thymidine, caused growth inhibition of SCVs. In turn, reversion of SCVs to NCVs was achieved by supplying extracellular dTMP. High-performance liquid chromatography additionally confirmed the intracellular lack of dTMP in SCVs, in contrast to NCVs. Moreover, the dTMP concentration is postulated to influence the intracellular persistence of S. aureus. Cell culture experiments with cystic fibrosis cells revealed that clinical and co-trimoxazole-induced SCVs with a diminished amount of dTMP showed significantly better intracellular persistence than NCVs. In conclusion, these results show that the dTMP concentration plays a key role in both the phenotypic appearance and the intracellular persistence of S. aureus.     

Steady-state staphylococcal enterotoxin type C mRNA is affected by a product of the accessory gene regulator (agr) and by glucose.

The effects of the accessory gene regulator (agr) and glucose on staphylococcal enterotoxin type C (SEC) gene (sec+) expression were examined. For the agr studies, a Tn551 insertionally inactivated agr was transferred into two different sec+ Staphylococcus aureus strains. Western blot (immunoblot) analysis showed that each of the sec+ Agr- derivatives produced less extracellular SEC than their Agr+ parent strains. Analysis of Northern (RNA) blots was consistent with at least part of the agr effect being at the level of steady-state sec+ mRNA. We examined the glucose effect on sec+ expression by utilizing both a fermentor system with a completely defined amino acid-containing medium in which the pH of the medium was maintained at 6.5 and a shake flask system with a complex medium in which the pH was allowed to fluctuate during bacterial growth. In both systems, samples from the cultures containing glucose had less extracellular SEC and less steady-state sec+ mRNA compared with the control cultures which lacked glucose. An intact agr was not required for the glucose effect on sec+ expression; MJB407, an Agr- sec+ strain, produced more SEC and had more steady-state sec+ mRNA when grown in medium that lacked glucose compared with medium that contained glucose.     

Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus

Thymidine-auxotrophic small colony variants (SCVs) of Staphylococcus aureus are frequently isolated from the chronically infected airways of patients suffering from cystic fibrosis. To date, little is known regarding the molecular mechanisms leading to the formation of this special phenotype, but the auxotrophism for thymidine suggests that impaired thymidine metabolism might play a major role. Sequence analysis of the thymidylate synthase-encoding thyA gene of six clinical thymidine-auxotrophic S. aureus SCVs revealed that all isolates had mutations within thyA. In five isolates the function of the thymidylate synthase was definitely impaired: three of them showed a truncation of the thyA coding sequence by nonsense or frame-shift mutations, in one further isolate the active site of the enzyme was affected by an internal 12-bp deletion, and another isolate had a 173-bp deletion spanning the 5'-terminal region of thyA and the preceding DNA sequence. The sixth isolate showed two amino acid substitutions within the thyA gene product. To confirm the importance of impaired thymidylate synthase synthesis or activity for the formation of the thymidine-auxotrophic SCV phenotype, we constructed a thyA knock-out mutant of a wild-type S. aureus strain. This mutant showed all characteristics of clinical SCVs, such as slow growth, decreased pigment production, reduced hemolytic activity, auxotrophism for thymidine, resistance to trimethoprim/sulfamethoxazol, and reduced plasma coagulase activity. Complementation of the thyA knock-out mutant with intact thyA in trans nearly restored the normal phenotype. In conclusion, these data confirm at the molecular level that impaired thymidylate synthase function is causative for the formation of the thymidine-auxotrophic SCV phenotype in S. aureus.     

Detection of Capsular Antigen Production in Unencapsulated Strains of Staphylococcus aureus

Of 91 compact-type strains of Staphylococcus aureus in regular serum soft agar (SSA), 82 converted diffuse-type growth in serum soft agar (pH adjusted to 6.0). With the addition of four different rabbit anticapsular sera (anti-type A, B, C, and D sera) in low pH (6.0) SSA, 21 strains of S. aureus showed compact-type colonial morphologies. Eleven, one, and one strains of S. aureus reacted singly with rabbit anticapsular sera types A, B, and C, respectively, and no strain reacted with rabbit anticapsular type D. Eight of the e strains reacted with both rabbit anticapsular sera types A and B. When the ability to absorb the converting activities of the antisera (changes of colonial morphologies of anticapsular sera in SSA) was quantitatively tested, 7- to 27-fold of these organisms were capable of absorbing the activities compared with the Smith diffuse organisms. These results suggest that even unencapsulated S. aureus strains are capable of producing capsular substance, although the capability is quantitatively different from strain to strain.