Coinfections and antimicrobial treatment in a cohort of falciparum malaria in a non-endemic country: a 10-year experience.

INTRODUCTION: Falciparum malaria remains one of the deadliest infectious diseases worldwide. In Germany, it is mainly an imported infection among travellers. Rates of coinfection are often unknown, and a clinical rationale for the beneficial use of calculated antibiotic therapy in patients with malaria and suspected coinfection is lacking. METHODS: We conducted an analysis of all in-patients treated with falciparum malaria at a German infectious diseases centre in vicinity to one of Europe's major airports for 2010-2019. Logistic regression and time-to-event analysis were used to evaluate predictors for bacterial coinfection, the use of antibacterial substances, as well as their influence on clinical course. RESULTS: In total, 264 patients were included. Of those, 64% received an additional antibacterial therapy (n = 169). Twenty-nine patients (11.0%) were found to have suffered from a relevant bacterial coinfection, while only a small fraction had relevant bacteremia (n = 3, 1.4%). However, patients with severe malaria did not suffer from coinfections more frequently (p = 0.283). CRP levels were not a reliable predictor for a bacterial coinfection (OR 0.99, 95% CI 0.94-1.06, p = 0.850), while another clinical focus of infection was positively associated (OR 3.86, 95% CI 1.45-11.55, p = 0.010). CONCLUSION: Although bacterial coinfections were rare in patients with malaria at our centre, the risk does not seem negligible. These data point rather towards individual risk assessment in respective patients than to general empiric antibiotic use.

Analysis of Mould Exposure of Immunosuppressed Patients at a German University Hospital.

Moulds are ubiquitous components of outdoor and indoor air and local conditions, temperature, humidity and season can influence their concentration in the air. The impact of these factors on mould exposure in hospitals and the resulting risk of infection for low to moderately immunocompromised patients is unclear. In the present retrospective analysis for the years 2018 to 2022, the monthly determined mould contamination of the outdoor and indoor air at the University Hospital Frankfurt am Main is compared with the average air temperature and the relative humidity. Mould infections (Aspergillus spp., Mucorales) of low to moderately immunosuppressed patients of a haematological-oncological normal ward were determined clinically according to the criteria of the European Organisation for Research and Treatment of Cancer (EORTC, Brussels, Belgium) and of the National Reference Centre for Surveillance of Nosocomial Infections (NRC-NI, Berlin, Germany). The data revealed that in the summer months (May-October), increased mould contamination was detectable in the outdoor and indoor air compared to the winter months (November-April). The mould levels in the patient rooms followed the detection rates of the outdoor air. Two nosocomial Aspergillus infections, one nosocomial Mucorales (Rhizopus spp.) infection (according to both NRC-NI and EORTC criteria) and five Aspergillus spp. infections (according to EORTC criteria) occurred in 4299 treated patients (resulting in 41,500 patient days). In our study, the incidence density rate of contracting a nosocomial mould infection (n = 3) was approximately 0.07 per 1000 patient days and appears to be negligible.

Seroprevalence of arthropod-borne bacterial infections in homeless individuals in Hamburg in 2020.

PURPOSE: The number of homeless people in Germany is steadily increasing. Due to their often precarious living conditions, this specific population may be increasingly exposed to ectoparasites that can transmit various pathogens. To assess the prevalence and thus the risk of such infections, we analyzed the seropositivity of rickettsiosis, Q fever, tularemia and bartonellosis in homeless individuals. METHODS: A total of 147 homeless adults from nine shelters in Hamburg, Germany, were included. The individuals underwent questionnaire-based interviewing, physical examination, and venous blood was drawn between May and June 2020. Blood samples were analyzed for antibodies against rickettsiae (Rickettsia typhi and R. conorii), Coxiella burnetii, Francisella tularensis and bartonellae. RESULTS AND CONCLUSION: A very low seroprevalence of R. typhi and F. tularensis infection was found (0-1%), while antibodies against R. conorii and C. burnetii were more common (7% each), followed by a relatively high seroprevalence of 14% for bartonellosis. Q fever seroprevalence was associated with the country of origin, whereas bartonellosis seroprevalence was associated with the duration of homelessness. Preventive measures targeting ectoparasites, especially body lice, should be put in place continuously.

Risk Factors and Role of Antibiotic Prophylaxis for Wound Infections after Percutaneous Endoscopic Gastrostomy.

BACKGROUND AND STUDY AIM: The incidence of wound infections after percutaneous endoscopic gastrostomy (PEG) varies widely in recent studies. The present study systematically investigates the underlying risk factors for the development of wound infections in a large cohort of patients over a long-term follow-up period. PATIENTS AND METHODS: A retrospective cohort study of patients undergoing PEG insertion using either the pull or push technique was conducted and patients followed up for 3 years. Tube-related wound infections were identified, and pathogens regularly cultured from wound swabs. Adjusted analysis was performed via univariate and multivariate logistic regression analysis. RESULTS: 616 patients were included in this study. A total of 25% percent of patients developed wound infections upon PEG tube insertion and 6.5% showed recurrent infections. Nicotine abuse (p = 0.01), previous ischemic stroke (p = 0.01) and head and neck cancer (p < 0.001) showed an increased risk for wound infection after PEG placement. Moreover, radio-chemotherapy was associated with the occurrence of wound infections (p < 0.001). Infection rates were similar between pull and push cohorts. The most common bacterial pathogen detected was Enterobacterales (19.2%). Staphylococcus aureus, Pseudomonas aeruginosa and enterococci were frequently detected in recurrent infection (14.2%, 11.4% and 9.6%, respectively). Antibiotic prophylaxis showed no effect on infection rates. CONCLUSIONS: Wound infections after PEG placement are common and occasionally occur as recurrent infections. There is potential for improvement in everyday clinical practice, particularly regarding antibiotic prophylaxis in accordance with guidelines.

First description of congenital toxoplasmosis after maternal coinfection with Toxoplasma gondii and severe acute respiratory syndrome coronavirus 2: a case report.

BACKGROUND: Congenital toxoplasmosis can be associated with serious clinical consequences from fetus to adulthood. Hence, early detection is required to minimize severe sequelae through appropriate therapy. We describe the first case of a congenital toxoplasmosis after maternal coinfection with Toxoplasma gondii and severe acute respiratory syndrome coronavirus 2 and the challenging serological diagnosis of the disease in this context. CASE PRESENTATION: A Caucasian boy was born at 27 weeks 2 days of gestation by cesarean section due to maternal COVID-19-related respiratory failure. Postpartum serological screening of the mother revealed a previously unrecognized active Toxoplasma gondii infection. The premature child initially tested negative for anti- Toxoplasma gondii immunoglobulin A and M antibodies 1, 2 and 4 weeks after birth, whereas immunoglobulin G antibodies were only weakly positive with no evidence of child-specific production. Neither neurological nor ophthalmological abnormalities were detected. Approximately 3 months after birth, serological testing indicated a congenital toxoplasmosis by presence of immunoglobulin A and M, in combination with a child-specific immunoglobulin G synthesis. Additionally, cerebrospinal fluid was tested positive for Toxoplasma gondii DNA. Although no clinical manifestations of congenital toxoplasmosis were detected, an antiparasitic therapy was initiated to minimize the risk of late sequelae. There were no hints for a transplacental transmission of severe acute respiratory syndrome coronavirus 2. CONCLUSION: This case raises the awareness of possible coinfections with the risk of transplacental transmission in cases of maternal coronavirus disease 2019. The report emphasizes the need for screening vulnerable patients for toxoplasmosis in general and especially in the context of pregnancy. It becomes evident that prematurity can complicate the serological diagnosis of congenital toxoplasmosis due to a delayed antibody response. Repeated testing is recommended to carefully monitor children at risk and especially those with a history of preterm birth.

Molecular surveillance of multidrug-resistant Gram-negative bacteria in Ukrainian patients, Germany, March to June 2022.

BackgroundSince the beginning of the war in Ukraine in February 2022, Ukrainians have been seeking shelter in other European countries.AimWe aimed to investigate the prevalence and the molecular epidemiology of multidrug-resistant Gram-negative (MDRGN) bacteria and meticillin-resistant Staphylococcus aureus (MRSA) in Ukrainian patients at admittance to the University Hospital Frankfurt, Germany.MethodsWe performed screening and observational analysis of all patients from March until June 2022. Genomes of MDRGN isolates were analysed for antimicrobial resistance, virulence genes and phylogenetic relatedness.ResultsWe included 103 patients (median age: 39 ±â€¯23.7 years), 57 of whom were female (55.3%; 95% confidence interval (CI): 45.2-5.1). Patients were most frequently admitted to the Department of Paediatrics (29/103; 28.2%; 95% CI: 19.7-37.9). We found 34 MDRGN isolates in 17 of 103 patients (16.5%; 95% CI: 9.9-25.1). Ten patients carried 21 carbapenem-resistant (CR) bacteria, five of them more than one CR isolate. Four of six patients with war-related injuries carried eight CR isolates. In six of 10 patients, CR isolates caused infections. Genomic characterisation revealed that the CR isolates harboured at least one carbapenemase gene, bla NDM-1 being the most frequent (n = 10). Core genome and plasmid analysis revealed no epidemiological connection between most of these isolates. Hypervirulence marker genes were found in five of six Klebsiella pneumoniae CR isolates. No MRSA was found.ConclusionHospitals should consider infection control strategies to cover the elevated prevalence of MDRGN bacteria in Ukrainian patients with war-related injuries and/or hospital pre-treatment and to prevent the spread of hypervirulent CR isolates.

Novel approaches for the serodiagnosis of louse-borne relapsing fever.

Louse-borne relapsing fever (LBRF) caused by B. recurrentis is a poverty-related and neglected infectious disease with an endemic focus in the Horn of Africa. Re-emergence of the disease occurred in Europe during the refugee crisis in 2015 and sporadic outbreaks were frequently reported in Eastern Africa where poor settings lack affordable diagnostics. Currently, there are no validated in vitro assays available for the serodiagnosis of LBRF. The aim of this study was to develop novel and reliable immunoassays by investigating clinically suspected and culture-confirmed serum samples from LBRF patients and a broad panel of serum samples from patients with other spirochetal, bacterial, and parasitic diseases. We identified two immunoreactive antigens (complement-inhibiting protein CihC and the glycerophosphodiester phosphodiesterase GlpQ of B. recurrentis) as the most promising target candidates leading to the evaluation of two immunoassays (line immunoblot and ELISA) for IgM and IgG. To optimize the IgM immunoassay, we conducted a bioinformatic approach to localize the relevant immunogenic regions within CihC. By utilizing a N-terminal CihC fragment, the sensitivity and specificity of both immunoassays (CihC and GlpQ) were high (IgM: sensitivity 100%, specificity of 89.9%, IgG: sensitivity 100%, specificity 99.2%). In conclusion, our findings indicate the diagnostic potential of CihC and GlpQ as valuable markers for the serodiagnosis of LBRF even at early time points of infection. Here, we provide strong evidence for the utilization of these immunoassays as reliable tools in clinical practice.

Anamnestic risk factor evaluation of patients carrying carbapenem-resistant Enterobacterales and/or Acinetobacter baumannii - impact on infection control management at a German University Hospital.

Background: Carbapenem-resistant Enterobacterales and Acinetobacter baumannii are of major concern in terms of infection prevention and control. This study evaluated factors that may increase the frequency of Enterobacterales and A. baumannii with carbapenem resistance (CR) in patients admitted to a German University Hospital for implementation of optimized infection control management. Methods: A five-year-retrospective epidemiological cohort analysis was conducted on anamnestic risk factors for carrying Enterobacterales and/or A. baumannii with CR in patients who were first tested positive for these species at University Hospital Frankfurt (UHF) between January 2013 and June 2018. Results: 364 patients were tested positive for Enterobacterales and/or A. baumannii with CR, resulting in n=400 bacterial isolates in total, with Klebsiella pneumoniae being the most frequently detected species (n=146/400; 36.5%; 95% confidence interval: 31.8-41.4). In patients who were tested positive for Enterobacterales and/or A. baumannii with CR, any hospital stay within the previous 12 months was the most frequently reported common factor (n=275/364; 75.5%; 70.8-79.9). Conclusion: A hospital stay within the previous 12 months, including hospitals in Germany and abroad, is a frequent characteristic of patients who tested positive for Enterobacterales and/or A. baumannii with CR. Upon admission, any previous hospital stay of the given patient within the previous 12 months should be determined. Infection control strategies such as screening measures need to be adapted to these patient groups in hospital settings. In order to reflect the varying determinants in "nosocomial" cases in greater detail, the existing criteria used to characterize "nosocomial detection" of gram-negative bacteria with CR should be reviewed.

Emergence of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae in vivo.

OBJECTIVES: The ß-lactam/ß-lactamase inhibitor combination ceftazidime/avibactam is active against KPC-producing Enterobacterales. Herein, we present molecular and phenotypic characterization of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae that emerged in vivo and in vitro. METHODS: Sequence analysis of blaKPC-3 was performed from clinical and in vitro-generated ceftazidime/avibactam-resistant K. pneumoniae isolates. Time-kill kinetics and the Galleria mellonella infection model were applied to evaluate the activity of ceftazidime/avibactam and imipenem alone and in combination. RESULTS: The ceftazidime/avibactam-resistant clinical K. pneumoniae isolate revealed the amino acid change D179Y in KPC-3. Sixteen novel mutational changes in KPC-3 among in vitro-selected ceftazidime/avibactam-resistant isolates were described. Time-kill kinetics showed the emergence of a resistant subpopulation under selection pressure with either imipenem or ceftazidime/avibactam. However, combined selection pressure with imipenem plus ceftazidime/avibactam prevented the development of resistance and resulted in bactericidal activity. Concordantly, the G. mellonella infection model revealed that monotherapy with ceftazidime/avibactam is prone to select for resistance in vivo and that combination therapy with imipenem results in significantly better survival. CONCLUSIONS: Ceftazidime/avibactam is a valuable antibiotic against MDR and carbapenem-resistant Enterobacterales. Based on time-kill kinetics as well as an in vivo infection model we postulate a combination therapy of ceftazidime/avibactam and imipenem as a strategy to prevent the development of ceftazidime/avibactam resistance in KPC-producing Enterobacterales in vivo.

Clinical impact of colonization with multidrug-resistant organisms on outcome after allogeneic stem cell transplantation in patients with acute myeloid leukemia.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment option for patients with acute myeloid leukemia (AML). During transplantation, patients undergo a period of severe neutropenia, which puts them at high risk for infectious complications. However, the impact of patient colonization with multidrug-resistant organisms (MDRO) on overall survival remains unclear. METHODS: In this retrospective, single-center study, the authors analyzed data from 264 patients with AML who underwent a first allo-HSCT between January 2006 and March 2016 at their institution. Primary endpoints were overall survival and nonrelapse-related mortality. RESULTS: One hundred forty-two of 264 patients (53.8%) were colonized by at least 1 MDRO, mainly with vancomycin-resistant Enterococcus faecalis/faecium (n = 122). The characteristics of colonized patients did not differ from those of MDRO-negative patients with respect to median age (53.5 vs 53 years), cytogenetic risk according to European LeukemiaNet criteria, remission status before allo-HSCT (first or second complete remission: 55.7% vs 60.7%, respectively; active disease: 44.4% vs 39.3%, respectively), donor type, or hematopoietic cell transplantation-comorbidity index (HCT-CI). Compared with noncolonized patients, MDRO-positive patients had an inferior probability of survival at 5 years (43.3% vs 65.5%; P = .002), primarily because of a higher cumulative incidence of nonrelapse-related mortality (33.9% vs 9.4%; P < .001). Death caused by infections occurred in 15.5% of colonized patients versus 4.9% of noncolonized patients. There was no difference in the cumulative incidence of relapse in MDRO-positive versus MDRO-negative patients (33.8% vs 42.1%, respectively; P = .798). CONCLUSIONS: The current data emphasize the importance of regular MDRO screenings and prompt further investigations into the impact of colonization with MDRO on the immune system after allo-HSCT. Cancer 2018;124:286-96. © 2017 American Cancer Society.

Multidrug-resistant bacteria in geriatric clinics, nursing homes, and ambulant care--prevalence and risk factors.

Colonization/infection with multidrug-resistant bacteria (MDRB) such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae, is an increasing problem not only in hospitals but also in long-term care facilities. The aim of this study was to determine the prevalence as well as the risk factors of colonization/infection with MRSA, VRE, and ESBL producing Enterobacteriaceae in geriatric clinics, nursing homes, and ambulant care in Frankfurt am Main, Germany. 288 patients from 2 geriatric clinics (n=46), 8 nursing homes (n=178), and 2 ambulant care facilities (n=64) as well as 64 staff members were screened for MDRB in the time period from October 2006 to May 2007. 58 patients (20.1%) and 4 staff members (6.2%) were colonized with MDRB. Among patients, 27 (9.4%) were colonized with MRSA, 11 (3.8%) were screened positive for VRE, and 25 (8.7%) were found to be colonized with ESBL producing Enterobacteriaceae. Prevalence of MDRB in geriatric clinics, nursing homes, and ambulant care facilities were 32.6%, 18.5%, and 15.6%, respectively. Significant risk factors for MDRB were immobility (OR: 2.7, 95% CI: 1.5-4.9; p=0.002), urinary catheter (OR: 3.1, 95% CI: 1.7-5.9; p<0.001), former hospitalization (OR: 2.1, 95% CI: 1.1-4.0; p=0.033), and wounds/decubiti (OR: 2.3, 95% CI: 1.5-4.9; p=0.03). Finally, the high level of MDRB in geriatric clinics, nursing homes, and ambulant care points to the importance of these institutions as a reservoir for dissemination.

Antimicrobial activities of trimethoprim/sulfamethoxazole, 5-iodo-2'-deoxyuridine and rifampicin against Staphylococcus aureus.

Trimethoprim/sulfamethoxazole (SXT), alone and in combination with rifampicin (RIF), is a therapeutic option against Staphylococcus aureus, including strains expressing meticillin resistance. However, the antimicrobial activity of SXT is antagonised by thymidine, which can be present in infected and/or inflamed tissues such as the airways of cystic fibrosis (CF) patients. In this study, thymidine concentrations in CF sputa were determined and the antimicrobial activities of SXT, 5-iodo-2'-deoxyuridine (IdUrd) and RIF alone and in combination against S. aureus were analysed. Thymidine concentrations in the sputa of ten different CF patients varied from <100 µg/L to 38845 µg/L. The abolished antimicrobial activity of SXT against 22 S. aureus strains in the presence of thymidine was restored by combination with IdUrd. In contrast, SXT combined with RIF in the presence of thymidine did not show a synergistic effect and, furthermore, allowed the emergence of RIF-resistant bacteria. Adding RIF to the combination of SXT and IdUrd did not improve antimicrobial activity against S. aureus. In conclusion, the combination of SXT and RIF as a therapeutic option against S. aureus infections in chronic inflamed tissues should be judged critically.

Multistep virtual screening for rapid and efficient identification of non-nucleoside bacterial thymidine kinase inhibitors.

Antimicrobial activity of trimethoprim/sulfamethoxazole (SXT) against Staphylococcus aureus (S. aureus) is antagonized by thymidine, which is abundant in infected or inflamed human tissue. To restore the antimicrobial activity of SXT in the presence of thymidine, we screened for small-molecule inhibitors of S. aureus thymidine kinase with non-nucleoside scaffolds. We present the successful application of an adaptive virtual screening protocol for novel antibiotics using a combination of ligand- and structure-based approaches. Two consecutive rounds of virtual screening and in vitro testing were performed that resulted in several non-nucleoside hits. The most potent compound exhibits substantial antimicrobial activity against both methicillin-resistant S. aureus strain ATCC 700699 and nonresistant strain ATCC 29213, when combined with SXT in the presence of thymidine. This study demonstrates how virtual screening can be used to guide hit finding in antibacterial screening campaigns with minimal experimental effort.

Synergistic antimicrobial activities of folic acid antagonists and nucleoside analogs.

The antimicrobial activities of folic acid antagonists are supposed to be antagonized by elevated extracellular thymidine concentrations in damaged host tissues. Therefore, this study was aimed at screening for nucleoside analogs that impair bacterial thymidine utilization and analyzing the combined antimicrobial activities of nucleoside analogs and folic acid antagonists in the presence of thymidine. Our screening results revealed that different nucleoside analogs, in particular halogenated derivatives of 2'-deoxyuridine, substantially impaired the bacterial utilization of extracellular thymidine in Staphylococcus aureus. Time-kill methods showed that 5-iodo-2'-deoxyuridine enhanced the extent of killing of trimethoprim-sulfamethoxazole (SXT) at 24 h against S. aureus in the presence of thymidine (200 microg/liter). While SXT (40 mg/liter) alone did not kill bacteria in the presence of thymidine, its combination with the nucleoside analog at a concentration of 8 mumol/liter showed a bactericidal effect. Moreover, 5-iodo-2'-deoxyuridine combined with SXT in the presence of thymidine showed a broad spectrum of activity against several Gram-positive and Gram-negative bacteria. In conclusion, these data provide evidence that the in vitro antimicrobial activity of SXT in the presence of thymidine can be significantly improved by combination with a nucleoside analog.

Mutational analyses of the BbCRASP-1 protein of Borrelia burgdorferi identify residues relevant for the architecture and binding of host complement regulators FHL-1 and factor H.

Borrelia burgdorferi exploits multiple strategies to evade host immune responses. One central immune escape mechanism is the inactivation of the host complement attack by acquisition host complement regulators FHL-1 and factor H via complement regulator-acquiring surface proteins (BbCRASPs). The BbCRASP-1 protein is the first bacterial factor H/FHL-1-binding protein for which the atomic structure has been solved. Previously, 3 regions including the C terminus were identified as putative contact sites for the two complement regulators by the pepspot analysis. Based on the crystallographic structure an in vitro mutagenesis approach was conducted to identify amino acid residues which are relevant for FHL-1 and factor H binding by exchanging single or multiple residues in region 1 and the C-terminally located region 3. Single changes at 4 positions in region 1 either reduced (Lys136, Lys141, Glu147) or completely eliminated (Leu146) binding of both complement regulators. Substitutions clustered within the C-terminal region decreased (Glu234, Lys238, Tyr239, Lys241, Asp244, Thr245) or abolished binding (Lys240, Asp242, Leu246) of both complement regulators. Mapping the mutations onto the atomic structure of BbCRASP-1 reveals that, in contrast to earlier assumption, the C-terminal mutations act indirectly on FHL-1 and factor H binding, whilst the region 1 mutations map the site of direct complement regulator interaction. The elucidation of BbCRASP-1 structure - function may allow development of novel therapeutic strategies against Lyme disease.

Thymidine-dependent Staphylococcus aureus small-colony variants: human pathogens that are relevant not only in cases of cystic fibrosis lung disease.

We report the isolation of thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus from unusual infection sites of patients with chronic soft tissue infection, tympanitis, bronchitis, peritonitis, and septicemia. Furthermore, we provide evidence that the essential growth factor for TD-SCVs, i.e., thymidine, and its metabolite dTMP are present in various human specimens.

The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates.

Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate > or = 10(-7)) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.

Linezolid resistance in Staphylococcus aureus: gene dosage effect, stability, fitness costs, and cross-resistances.

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

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.