Factors associated with nonhome discharge after endovascular aneurysm repair.

OBJECTIVE: This study aims to identify preoperative factors associated with nonhome discharge (NHD) after endovascular aneurysm repair (EVAR). NHD has implications for patient care, readmission, and long-term mortality; nevertheless, the existing literature lacks information regarding factors associated with NHD for patients undergoing EVAR. In contrast, our study assesses preoperative factors associated with NHD for this population by using national data from the Vascular Quality Initiative. METHODS: We identified adult patients who underwent elective EVAR in the Vascular Quality Initiative (2003-2022) and excluded those who were not living at home preoperatively. Multivariable logistic regression was used to identify preoperative factors associated with NHD. Kaplan-Meier methods and Cox-regression analyses were used to assess the impact of NHD on 5-year survival as a secondary outcome. RESULTS: We included 61,792 patients, of which 3155 (5.1%) had NHD. NHD patients were more likely to be older (79 years [interquartile range, 73-18 years] vs 73 years [interquartile range, 67-79 years]), female (33.7% vs 18.2%; P < .001), non-White (16.0% vs 11.7%; P < .001), and have more comorbidities. NHD patients had higher rates of postoperative complications (acute kidney injury, 11.9% vs 2.0% [P < .001]; myocardial infarction, 3.8% vs 0.5% [P < .001]; and in-hospital reintervention, 4.7% vs 0.5% [P = .033]). Multivariable analysis revealed many preoperative characteristics were associated with higher odds of NHD: most notably, age (per additional decade: odds ratio [OR], 2.15; 95% confidence interval [CI], 2.03-2.28; P < .001), female sex (OR, 1.79; 95% CI, 1.63-1.95; P < .001) and aneurysm diameter >65 mm (OR, 2.18; 95% CI, 1.98-2.39; P < .001), along with potentially modifiable factors, including anemia, chronic obstructive pulmonary disease, chronic heart failure, weight, and diabetes. In contrast, aspirin, statin, and angiotensin-converting enzyme inhibitor/angiotensin II receptor blocekr use were associated with lower odds of NHD. NHD was associated with higher hazards of 5-year mortality, even after adjusting for confounders (40% vs 14%; adjusted hazard ratio, 2.13; 95% CI, 1.86-2.44; P < .001). CONCLUSIONS: Several factors were associated with higher odds of NHD after elective EVAR, including nonmodifiable factors such as female sex and larger aortic diameter, and potentially modifiable factors such as anemia, chronic obstructive pulmonary disease, chronic heart failure, body mass index, and diabetes. Special attention should be given to populations with nonmodifiable factors, and efforts at optimizing medical conditions with higher NHD likelihood seems appropriate to improve patient outcomes and quality of life after EVAR.

Femoral vein translocation for complex hemodialysis access.

OBJECTIVE: Vascular access is difficult in patients without suitable arm veins and prior graft infections. The use of femoral vein translocation (FVT) to the upper extremity or contralateral lower extremity for hemodialysis access may be associated with low infection rates and high patency rates. FVT is used for patients without central venous occlusion who have failed prior access either owing to graft infection or repetitive graft thrombosis. The largest case series consists of 30 cases. The objective of this study is to determine the infection incidence, primary patency, primary-assisted patency, and secondary patency rates among FVTs. METHODS: A retrospective chart review was performed on all patients who underwent FVT by a single vascular practice over a 10-year period (2013-2023). Study variables included length to last follow-up (months), prior access, prior graft infection, comorbid conditions, primary patency, primary-assisted patency, secondary patency, postoperative steal syndrome, postoperative graft infection, postoperative harvest site complication, and postoperative lower extremity compartment syndrome. RESULTS: A total of 131 FVTs were performed from 2013 to 2023; 126 patients (47% male, 53% female; 76% Black, 24% White) with a mean age of 52 ± 14 years and a mean body mass index of 29 ± 8 had at least 1 month of follow-up and were included for analysis. The median follow-up was 46 months (interqurtile range, 19-72 months). The mean number of prior permanent accesses was 2.5 ± 1.4. Forty-eight percent of patients had prior graft infections. The primary, primary-assisted, and secondary patency rates were 66%, 93%, and 98%, respectively, at 6 months; 43%, 85%, and 96% at 12 months; 25%, 70%, and 92% at 24 months; 16%, 61%, and 88% at 36 months; and 14%, 56%, and 82% at 48 months. Postoperative steal syndrome and postoperative access infection requiring excision was observed 16% and 5% of patients, respectively. Harvest site complications requiring an additional procedure occurred in 19% of cases. Three patients developed lower extremity compartment syndrome postoperatively, requiring fasciotomy. Six patients developed chronic lower extremity edema after femoral vein harvest. The mean procedure time and hospital length of stay were 197 ± 40 minutes and 3.5 ± 2.8 days, respectively. CONCLUSIONS: FVT is associated with low infection rates and high long-term patency rates. Significant postoperative complications include steal syndrome and harvest site complications. FVT remains a viable option for patients who have failed prior access owing to graft infection or repetitive graft thrombosis.

Locoregional anesthesia is associated with reduced hospital stay and need for intensive care unit care of elective endovascular aneurysm repair patients in the Vascular Quality Initiative.

OBJECTIVE: It has been shown local or regional anesthetic techniques are a feasible alternative to general anesthesia for endovascular aortic aneurysm repair (EVAR). However, studies to date have shown controversial findings with respect to the benefit of locoregional anesthesia (LR) in the elective setting. The objective of this study is to compare postoperative outcomes between LR and general anesthesia (GA) in the setting of elective EVAR, using a large, multicenter database. METHODS: Using the Society for Vascular Surgery Vascular Quality Initiative database, we retrospectively analyzed all patients who underwent elective EVAR from August 2003 to June 2021. Patients were grouped by anesthetic type based on the level of consciousness afforded by the anesthetic: local or regional anesthesia (LR) vs GA. Primary outcomes were total postoperative hospital length-of-stay (LOS) and intensive care unit (ICU) LOS. Propensity score matching was used for risk adjustment and to analyze the primary outcomes with confirmatory analysis using logistic or linear regression, as appropriate, in single and multilevel models. Secondary outcomes were 30-day mortality, 1-year mortality, postoperative outcomes, operative time, fluoroscopy time, and reoperation rate. These were analyzed following propensity score matching as well as using logistic regression and Cox proportional hazard regression in single and multilevel models, as appropriate. RESULTS: A total of 50,809 patients underwent elective EVAR from 2003 to 2021. Of these, 4302 repairs used LR (8.5%) and 46,507 (91.5%) were performed under GA. After employing propensity score matching, two groups of 3027 patients were produced. These showed no significant difference in 30-day mortality (odds ratio, 1.22; P = .53), 1-year mortality (hazard ratio, 1.06; P = .62), or any postoperative outcomes. LR was found to be significantly associated with shorter hospital stays (≤2 days) (12.5% vs 14.8%; P = .01), decreased ICU utilization (19.3% vs 30.6%; P < .001), decreased operative time (110.8 vs 117.3 minutes; P < .001), decreased fluoroscopy time (21.0 vs 22.7 minutes; P < .001), and a slight reduction in reoperation rate (1.2% vs 1.9%; P = .02), which all remained significant following single-level and multilevel multivariate analyses accounting for hospital and physician random effects. CONCLUSIONS: These data suggest that LR anesthesia is safe and may offer advantages in reducing resource utilization for patients undergoing elective EVAR, primarily based on associations with reduced ICU care and reduced hospital stay. Given these findings, LR may prove an advantageous technique in appropriately selected patient populations.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

Predictors of blunt abdominal aortic injury in trauma patients and mortality analysis.

OBJECTIVE: Blunt abdominal aortic injury (BAAI) occurs in less than 0.1% of blunt traumas. A previous multi-institutional study found an associated mortality rate of 39%. We sought to identify risk factors for BAAI and risk factors for mortality in patients with BAAI using a large national database. We hypothesized that an Injury Severity Score of 25 or greater, and thoracic trauma would both increase the risk of mortality in patients with BAAI. METHODS: The Trauma Quality Improvement Program (2010-2016) was queried for individuals with blunt trauma. Patients with and without BAAI were compared. Covariates were included in a multivariable logistic regression model to determine mechanisms of injury, examination findings, and concomitant injuries associated with increased risk for BAAI. An additional multivariable analysis was performed for mortality in patients with BAAI. RESULTS: From 1,056,633 blunt trauma admissions, 1012 (0.1%) had BAAI. The most common mechanism of injury was motor vehicle accident (MVA; 57.5%). More than one-half the patients had at least one rib fracture (54.0%), or a spine fracture (53.9%), whereas 20.8% had hypotension on admission and 7.8% had a trunk abrasion. The average length of stay was 13.4 days and 24.6% required laparotomy, with 6.6% receiving an endovascular repair and 2.9% an open repair. The risk of death in those treated with endovascular vs open repair was similar (P = .28). On multivariable analysis, MVA was the mechanism associated with the highest risk of BAAI (odds ratio [OR], 4.68; 95% confidence interval [CI], 3.87-5.65; P < .001) followed by pedestrian struck (OR, 4.54; 95% CI, 3.47-5.92; P < .001). Other factors associated with BAAI included hypotension on admission (OR, 3.87; 95% CI, 3.21-4.66; P < .001), hemopneumothorax (OR, 3.67; 95% CI, 1.16-11.58; P < .001), abrasion to the trunk (OR, 1.49; 95% CI, 1.15-1.94; P = .003), and rib fracture (OR, 1.46; 95% CI, 1.25-1.70; P < .001). The overall mortality rate was 28.0%. Of the variables examined, the strongest risk factor associated with mortality in patients with BAAI was hemopneumothorax (OR, 12.49; 95% CI, 1.25-124.84; P = .03) followed by inferior vena cava (IVC) injury (OR, 12.05; 95% CI, 2.80-51.80; P < .001). CONCLUSIONS: In the largest nationwide series to date, BAAI continues to have a high mortality rate with hemopneumothorax and IVC injury associated with the highest risk for mortality. The mechanism most strongly associated with BAAI is MVA followed by pedestrian struck. Other risk factors for BAAI include rib fracture and trunk abrasion. Providers must maintain a high suspicion of injury for BAAI when these mechanisms of injury, physical examination or imaging findings are encountered.

Staphylococcus aureus in the airways of cystic fibrosis patients - A retrospective long-term study.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disease associated with chronic airway infections by Staphylococcus aureus as one of the earliest and most prevalent pathogens. We conducted a retrospective study to determine the S. aureus infection status of CF patients treated since 1994 at two certified CF-centres in Münster, Germany, to get insights into the dynamics of S. aureus airway infection and the clinical impact on lung function on a long-term perspective. MATERIALS AND METHODS: We used data from our microbiological database collected between 1994 and 2016 for patients treated at two centres in Münster, Germany, respectively, to determine the infection status for S. aureus. Furthermore, the resistance to selected antibiotics was determined for all patients' isolates and for 15 patients on a longitudinal basis. In addition, the prevalence of adaptive phenotypes such as small colony variants (SCVs) and mucoid S. aureus was assessed. RESULTS: For this study, 2867 patient years with respiratory specimens (mean of 9.3 years for every patient, range 1-22 years) were evaluated for 283 CF patients (median age of 7 years at the beginning of the observation period, range 0-57 years, 51% male). 18% of patients were rarely infected by S. aureus (≤24% of observation years), 20% of patients intermittently (25-49%) and 61% persistently (≥50% of observation period). Susceptibility testing for 12969 S. aureus isolates resulted in resistance to methicillin in 9%, trimethoprim/sulfamethoxazole in 10%, levofloxacin in 14%, gentamicin in 20%, erythromycin and/or clindamycin in 30% and penicillin in 80% of all isolates. S. aureus isolates of 15 patients revealed dynamics of resistance with increase, decrease and loss of resistant isolates to the analysed antibiotics during the study period. SCVs were isolated at least once from 42% (n = 118) of patients and mucoid isolates from 2% (n = 7) of patients. In the last study year, 89 patients were infected by S. aureus only, 44 patients by S. aureus and Pseudomonas aeruginosa and 18 by P. aeruginosa only. Patients infected by S. aureus only were younger and had better lung function compared to the other two groups. CONCLUSIONS: We determined a high percentage of patients with persistent S. aureus infection. During persistence, mostly fluctuation of resistance against various antibiotics was observed in the isolates indicating acquisition and loss of resistance genes by S. aureus. The prevalence of adaptive phenotypes during long-term persistence was high for SCVs (42% of patients), but low for mucoid isolates (2% of patients), which might be underestimated for mucoid phenotypes due to the retrospective study design and the difficulty to detect mucoid isolates in primary cultures. While patients with S. aureus only had better lung function and were younger, no difference was found between the group of P. aeruginosa and S. aureus co-infection and P. aeruginosa only with previous S. aureus infection.

Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus.

Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256 When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

Biological characterization of lipoic acid- and heme-dependent Escherichia coli small colony variants isolated from sheep in Xinjiang, China.

Escherichia coli (E. coli) small colony variants (SCVs) have garnered attention due to their heightened antibiotic resistance and enhanced cell retention, posing significant risks to public health and food safety. However, understanding of SCVs derived from sheep remains limited. This study aimed to detect the biological characterization of sheep-derived E. coli SCVs and investigate the factors contributing to SCV development with preliminary genomic data. In this study, a lipoic acid-dependent SCV (LA-SCV) and a wild-type (WT) strain were isolated from sheep bile. Then, a heme-dependent SCV (HD-SCV) was induced from WT using amikacin. Initially, we examined factors contributing to SCV formation via comparative genomics. Subsequent comparisons between WT and two SCV strains encompassed antibiotic resistance, hemolytic activity, biofilm formation, motility, and metabolism. Genomic analyses identified a frameshift deletion mutation in the lipA gene in LA-SCV and a stopgain mutation in the hemG gene in HD-SCV, hypothesized as potential triggers for lipoic acid- and heme-dependent SCV development, respectively. Physiological, biochemical, and cultural traits exhibited notable differences between WT and SCVs, including increased antibiotic resistance, hemolytic activity, and biofilm formation, but alongside non-fermentative acetate utilization, slow growth, reduced intracellular ATP, and decreased motility (P < 0.01). The energy and amino acid metabolism were suppressed during the logarithmic phase in LA-SCV, while both logarithmic and stable phases in HD-SCV. These alterations in biological characteristics present significant challenges in managing E. coli pathogenicity and antibiotic resistance.

Deciphering the induction of Listeria monocytogenes into sublethal injury using fluorescence microscopy and RT-qPCR.

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA-/PI+ cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA-/PI-) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA+). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment.

Unravelling staphylococcal small-colony variants in cardiac implantable electronic device infections: clinical characteristics, management, and genomic insights.

Objectives: Staphylococcal small-colony variants (SCVs) are common in cardiac implantable electronic device (CIED) infections. This is the first retrospective and multi-case study on CIED infections due to staphylococcal SCVs, aiming to provide a theoretical basis for the clinical management of CIED and device-related infections caused by staphylococcal SCVs. Methods: Ninety patients with culture positive CIED infections were enrolled between 2021 and 2022. We compared the demographic and clinical characteristics of patients with and without SCVs and performed genomic studies on SCVs isolates. Results: Compared to patients without SCVs, those with SCVs had a longer primary pacemaker implantation time and were more likely to have a history of device replacement and infection. They showed upregulated inflammatory indicators, especially higher NEUT% (52.6 vs. 26.8%, P = 0.032) and they had longer hospital stays (median 13 vs. 12 days, P = 0.012). Comparative genomics analysis was performed on Staphylococcus epidermidis wild-type and SCVs. Some genes were identified, including aap, genes encoding adhesin, CHAP domain-containing protein, LPXTG cell wall anchor domain-containing protein, and YSIRK-type signal peptide-containing protein. Conclusion: Staphylococcal SCVs affect the clinical characteristics of CIED infections. The process of staphylococcal SCVs adherence, biofilm formation, and interaction with neutrophils play a vital role.

Emergence of Thymidine-Dependent Staphylococcus aureus Small-Colony Variants in Cystic Fibrosis Patients in Southern Brazil.

We characterized Staphylococcus aureus small-colony variant (SCV) strains isolated from cystic fibrosis (CF) patients in southern Brazil. Smaller colonies of S. aureus were isolated from respiratory samples collected consecutively from 225 CF patients from July 2013 to November 2016. Two phenotypic methods-the auxotrophic classification and a modified method of antimicrobial susceptibility testing-were employed. PCR was conducted to detect the mecA, ermA, ermB, ermC, msrA, and msrB resistance genes. Furthermore, DNA sequencing was performed to determine the mutations in the thyA gene, and multilocus sequence typing was used to identify the genetic relatedness. S. aureus strains were isolated from 186 patients (82%); suggestive colonies of SCVs were obtained in 16 patients (8.6%). The clones CC1 (ST1, ST188, and ST2383), CC5 (ST5 and ST221), and ST398 were identified. Among SCVs, antimicrobial susceptibility testing showed that 77.7% of the isolates were resistant to multiple drugs, and all of them were susceptible to vancomycin. mecA (2), ermA (1), ermB (1), ermC (3), and msrB (18) were distributed among the isolates. Phenotypically thymidine-dependent isolates had different mutations in the thyA gene, and frameshift mutations were frequently observed. Of note, revertants showed nonconservative or conservative missense mutations. SCVs are rarely identified in routine laboratory tests. IMPORTANCE Similar findings have not yet been reported in Brazil, emphasizing the importance of monitoring small-colony variants (SCVs). Altogether, our results highlight the need to improve detection methods and review antimicrobial therapy protocols in cystic fibrosis (CF) patients.

Q Fever: Seroprevalence, Risk Factors in Slaughter Livestock and Genotypes of Coxiella burnetii in South Africa.

Q fever is a neglected zoonosis in South Africa, causing significant losses in livestock and game animals through reproductive disorders. However, there are limited studies on the extent of Coxiella burnetii infections in livestock in South Africa. Further, there is also lack of knowledge about the types of C. burnetii strains that are currently circulating in the country. Therefore, a cross-sectional, abattoir-based study was conducted to determine the seroprevalence of C. burnetii and associated risk factors, and to characterize C. burnetii strains from slaughter livestock at red meat abattoirs in Gauteng, South Africa. Of the 507 animals tested, 6.9% (95% CI: 4.9-9.5%) were positive for antibodies against C. burnetii. The seroprevalence was 9.4% (31/331) in cattle, 4.3% (3/69) in sheep, and 0.9% (1/107) in pigs. Out of the 63 tissue samples from 35 seropositive animals including material from two sheep aborted fetuses from Mangaung district (Free State province), 12.7% (8/63) tested positive by IS1111 PCR. Genotyping of the eight PCR-positive tissues from eight animals by MLVA revealed two novel genotypes, not available in Coxiella MLVA databases. It is concluded that slaughter animals pose a risk of exposing abattoir and farm workers to C. burnetii in South Africa.

Mechanisms of Antibiotic Failure During Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is a highly successful Gram-positive pathogen capable of causing both superficial and invasive, life-threatening diseases. Of the invasive disease manifestations, osteomyelitis or infection of bone, is one of the most prevalent, with S. aureus serving as the most common etiologic agent. Treatment of osteomyelitis is arduous, and is made more difficult by the widespread emergence of antimicrobial resistant strains, the capacity of staphylococci to exhibit tolerance to antibiotics despite originating from a genetically susceptible background, and the significant bone remodeling and destruction that accompanies infection. As a result, there is a need for a better understanding of the factors that lead to antibiotic failure in invasive staphylococcal infections such as osteomyelitis. In this review article, we discuss the different non-resistance mechanisms of antibiotic failure in S. aureus. We focus on how bacterial niche and destructive tissue remodeling impact antibiotic efficacy, the significance of biofilm formation in promoting antibiotic tolerance and persister cell formation, metabolically quiescent small colony variants (SCVs), and potential antibiotic-protected reservoirs within the substructure of bone.

Thymidine-auxotrophic Staphylococcus aureus small-colony variant bacteremia in a patient with cystic fibrosis.

BACKGROUND: Small-colony variants (SCVs) are a morphologic subtype of Staphylococcus aureus that may occur through several mechanisms including auxotrophism for thymidine, hemin, or menadione. Auxotrophic SCV for thymidine fail to synthesize DNA specifically because of mutations in the thymidylate synthase gene. We isolated S. aureus thymidine-dependent SCVs (TD-SCV) from blood and respiratory samples of a pediatric patient with cystic fibrosis and pulmonary exacerbation. METHODS: Nutritional dependence of SCVs on hemin, menadione, and thymidine was evaluated. Antimicrobial susceptibility testing was performed through broth microdilution. Polymerase chain reaction was carried out for mecA, ermA, ermB, ermC, msrA, and msrB resistance genes. DNA sequencing was used to determine mutations in thyA and the multilocus sequence typing to identify genetic relatedness. RESULTS: Methicillin-sensitive S. aureus with normal and TD-SCV phenotypes were isolated from respiratory samples and a TD-SCV phenotype was isolated from blood culture. Macrolides resistance was attributed to ermC and msrB genes. All isolates belonged to ST398. The thyA gene in S. aureus is 957 nucleotides in length and encodes a protein of 318 amino acids. The TD-SCV isolates carried a -2 nt frameshift mutation (delta 667GC668) in thyA, creating a stop codon at residue 222 close to the predicted binding site for deoxyuridine monophosphate. CONCLUSIONS: The pathogenesis of SCVs is complex and not fully elucidated. Factors inherent to the patient such as physiological conditions, recurrent infections, or coinfection should be considered. Although SCVs are considered less virulent, they showed the ability to invade and cause bacteremia in the patient.

An effective weapon against biofilm consortia and small colony variants of MRSA.

OBJECTIVES: This study was designed to investigate the effect of AgNPs (10 nm and 30 nm) on different phenotypes of Staphylococcus aureus biofilm consortia. MATERIALS AND METHODS: A total of eighteen biofilm-producing isolates of Methicillin-Resistant S. aureus (MRSA) were used in the present study. Tube methods, Congo-red agar method, and scanning electron microscopy (SEM) were used to study biofilm phenotypes. Population analysis assay on a tryptone soya agar (TSA) plate was applied to study the different phenotypes of biofilm consortia. The effect of AgNPs was evaluated by broth dilution assay. RESULTS: Results showed that biofilm consortia harbour different phenotypes, i.e., planktonic, metabolically inactive cells, and small colony variants (SCVs) or persister cells. The focus of the present study is the effect of AgNPs on biofilm consortia of MRSA, particularly on the SCVs population. Large size AgNPs (30 nm) were unable to diffuse through extracellular matrix material coverings of the biofilm consortia; they were only active against the planktonic population that occupies the outer surface of consortia. The smaller AgNPs (10 nm), on the other hand, were found to diffuse through the matrix material and hence were effective against planktonic as well as metabolically inactive population of consortia. Moreover, 30 nm AgNPs take 6 hr to disperse off and kill planktonic and upper surface indwellers. The 10 nm AgNPs disperse and kill the majority of biofilm indwellers within 20 min. CONCLUSION: The present study showed that 10 nm AgNPs can easily penetrate inside the biofilm and are active against all of the indwellers of consortia.

Consequences of Metabolic Interactions during Staphylococcus aureus Infection.

Staphylococcus aureus is a metabolically flexible pathogen that causes infection in diverse settings. An array of virulence factors, including the secreted toxins, enables S. aureus to colonize different environmental niches and initiate infections by any of several discrete pathways. During these infections, both S. aureus and host cells compete with each other for nutrients and remodel their metabolism for survival. This metabolic interaction/crosstalk determines the outcome of the infection. The reprogramming of metabolic pathways in host immune cells not only generates adenosine triphosphate (ATP) to meet the cellular energy requirements during the infection process but also activates antimicrobial responses for eventual bacterial clearance, including cell death pathways. The selective pressure exerted by host immune cells leads to the emergence of bacterial mutants adapted for chronicity. These host-adapted mutants are often characterized by substantial changes in the expression of their own metabolic genes, or by mutations in genes involved in metabolism and biofilm formation. Host-adapted S. aureus can rewire or benefit from the metabolic activities of the immune cells via several mechanisms to cause persistent infection. In this review, we discuss how S. aureus activates host innate immune signaling, which results in an immune metabolic pressure that shapes S. aureus metabolic adaptation and determines the outcome of the infection.

Rifampicin-Loaded Mesoporous Silica Nanoparticles for the Treatment of Intracellular Infections.

Infectious diseases remain a major burden in today's world, causing high mortality rates and significant economic losses, with >9 million deaths per year predicted by 2030. Invasion of host cells by intracellular bacteria poses treatment challenges due to the poor permeation of antimicrobials into the infected cells. To overcome these limitations, mesoporous silica nanoparticles (MSNP) loaded with the antibiotic rifampicin were investigated as a nanocarrier system for the treatment of intracellular bacterial infection with specific interest in the influence of particle size on treatment efficiency. An intracellular infection model was established using small colony variants (SCV) of S. aureus in macrophages to systemically evaluate the efficacy of rifampicin-loaded MSNP against the pathogen as compared to a rifampicin solution. As hypothesized, the superior uptake of MSNP by macrophages resulted in an enhanced treatment efficacy of the encapsulated rifampicin as compared to free antibiotic. This study provides a potential platform to improve the performance of currently available antibiotics against intracellular infections.

The Energy-Coupling Factor Transporter Module EcfAA'T, a Novel Candidate for the Genetic Basis of Fatty Acid-Auxotrophic Small-Colony Variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA'T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA'T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

Staphylococcus aureus Regulator Sigma B is Important to Develop Chronic Infections in Hematogenous Murine Osteomyelitis Model.

Staphylococcus aureus is a major pathogen causing bone infections that can become chronic and difficult to treat. Recently, we described the mechanism employed by S. aureus to switch to small colony variants (SCVs) and trigger intracellular bacterial persistence through the global stress regulator SigB. Here, we studied the role of SigB in the formation of chronic osteomyelitis. We used a murine hematogenous osteomyelitis model, where the mice were infected via the tail vein and subsequently developed chronic osteomyelitis. Mice were infected with S. aureus LS1, LS1ΔsigB and LS1ΔsigB complemented and kidney and bone tissues were analyzed six weeks after infection. S. aureus LS1ΔsigB formed a high rate of abscesses in kidneys, but the bacterial loads and the weight loss of the animals were lower in comparison with animals infected with the wild type and the complemented strain, indicating a more rapid and efficient bacterial clearing by the host immune system. Moreover, the sigB-mutant was not able to form SCV phenotypes either in kidney or in bone tissue. Our results demonstrate that staphylococcal SigB is important to avoid bacterial elimination by the host immune response, establish a bone infection and mediate bacterial adaptation (SCV-formation) for persistent infections.

Addition of thymidine to culture media for accurate examination of thymidine-dependent small-colony variants of methicillin-resistant Staphylococcus aureus: a pilot study.

Small-colony variants (SCVs) are slow-growing subpopulations of various auxotrophic bacterial strains. Thymidine-dependent SCVs (TD-SCVs) are unable to synthesize thymidine; hence, these variants fail to grow in a medium without thymidine. In this study, we used 10 TD-SCVs of Staphylococcus aureus, of which four strains possessed mecA. We compared the efficacy of a newly modified medium containing thymidine for the detection of TD-SCVs of methicillin-resistant S. aureus (MRSA) to the efficacy of routinely used laboratory media. We observed that none of the 10 TD-SCVs of S. aureus grew in Mueller-Hinton agar, and four TD-SCVs of MRSA failed to grow on all MRSA screening media, except for the ChromID™ MRSA medium. Laboratory tests conducted using medium with thymidine incorporated showed that thymidine did not affect the minimum inhibitory concentrations of oxacillin and cefoxitin for clinical isolates of S. aureus, and was able to detect MRSA, including TD-SCVs. These findings showed that thymidine-incorporated media are able to detect TD-SCVs of MRSA without altering the properties of other clinically isolated MRSA strains.