Optimized decision algorithm for the microbiological diagnosis of osteoarticular infections in adults using synovial fluid samples: a prospective study in two French hospitals including 423 samples of synovial fluid.

No consensus exists about the techniques to use for microbiological diagnosis of bone and joint infections (BJIs). The objective herein was to define an algorithm to optimize BJI diagnosis in adults using various bacteriological methods on synovial fluid samples. This prospective multi-center study included 423 synovial fluids collected from adult patients with suspected BJIs. Culture (using five solid media, an enrichment broth, and blood culture bottles), universal 16S rRNA PCR followed by Sanger sequencing, and seven specific bacterial PCRs were systematically performed. Combinations of methods were compared to arrive at the optimized algorithm. Among 423 synovial fluids, 242 infections were diagnosed (57.2 %): 213 mono- and 29 poly-microbial for a total of 284 bacteria (staphylococci at 54.6 %, streptococci-enterococci at 16.5 %, Gram-negative bacilli at 15.5 %, anaerobic species at 8.8 %). Comparing culture techniques, blood culture bottles had the highest sensitivity (67.6 % for pediatric and 63.9 % for anaerobic bottles) but are not sufficient alone and require being combined with solid media. The 16S rDNA PCR detected only 52.3 % of the bacteria, whereas specific PCRs had a higher sensitivity (Staphylococcus spp. at 66.2 %, S. aureus at 85.2 %, Streptococcus spp. at 91.2 %). Based on these results, an algorithm was proposed associating three solid media; inoculation into blood culture bottles; and 16S, Staphylococcus spp., and Streptococcus spp. PCRs, which would have detected 90.5 % of bacteria in the present cohort versus 79.2 % using all culture techniques on synovial fluid. This prospective study shows that a combination of culture and molecular methods on synovial fluids allows the optimization of bacterial detection.

Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study.

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis.

OBJECTIVES: To assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. METHODS: The study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. RESULTS: Only one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8-89.8%) and the negative percentage agreement was 100% (95% CI, 97.2-100%). The positive predictive value was extremely high (100%; 95% CI, 97.6-100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7-88.2%), partially explained by the missing target species in the panel. DISCUSSION: The BF-JIP showed high performances to detect pathogens involved in acute arthritis.

Staphylococcus succinus Infective Endocarditis, France.

Infective endocarditis is a rare condition in humans and is associated with high illness and death rates. We describe a case of infective endocarditis caused by Staphylococcus succinus bacteria in France. We used several techniques for susceptibility testing for this case to determine the oxacillin profile.

Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

LipoParticles: a lipid membrane coating onto polymer particles to enhance the internalization in osteoblast cells.

LipoParticles, core-shell assemblies consisting of a polymer core coated by a lipid membrane, are promising carriers for drug delivery applications with intracellular targets. This is of great interest since it is actually challenging to treat infections involving intracellular bacteria such as bone and joint infections where the bacteria are hidden in osteoblast cells. The present work reports for the first time to the best of our knowledge the proof of enhanced internalization of particles in osteoblast cells thanks to a lipid coating of particles (= LipoParticles). The ca. 300 nm-sized assemblies were elaborated by reorganization of liposomes (composed of DPPC/DPTAP 10/90 mol/mol) onto the surface of poly(lactic-co-glycolic acid) (PLGA) particles, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zetametry. Optimization of these assemblies was also performed by adding poly(ethylene glycol) (PEG) chains on their surface (corresponding to a final formulation of DPPC/DPTAP/DPPE-PEG5000 8/90/2 mol/mol/mol). Interestingly, this provided them colloidal stability after their 20-fold dilution in PBS or cell culture medium, and made possible their freeze-drying without forming aggregates after their re-hydration. Their non-cytotoxicity towards a human osteoblast cell line (MG63) was also demonstrated. The enhanced internalization of LipoParticles in this MG63 cell line, in comparison with PLGA particles, was proven by observations with a confocal laser scanning microscope, as well as by flow cytometry assays. Finally, this efficient internalization of LipoParticles in MG63 cells was confirmed by TEM on ultrathin sections, which also revealed localization close to intracellular Staphylococcus aureus.

Multicenter evaluation of the BIOFIRE Joint Infection Panel for the detection of bacteria, yeast, and AMR genes in synovial fluid samples.

The bioMérieux BIOFIRE Joint Infection (JI) Panel is a multiplex in vitro diagnostic test for the simultaneous and rapid (~1 h) detection of 39 potential pathogens and antimicrobial resistance (AMR) genes directly from synovial fluid (SF) samples. Thirty-one species or groups of microorganisms are included in the kit, as well as several AMR genes. This study, performed to evaluate the BIOFIRE JI Panel for regulatory clearance, provides data from a multicenter evaluation of 1,544 prospectively collected residual SF samples with performance compared to standard-of-care (SOC) culture for organisms or polymerase chain reaction (PCR) and sequencing for AMR genes. The BIOFIRE JI Panel demonstrated a sensitivity of 90.9% or greater for all but six organisms and a positive percent agreement (PPA) of 100% for all AMR genes. The BIOFIRE JI Panel demonstrated a specificity of 98.5% or greater for detection of all organisms and a negative percent agreement (NPA) of 95.7% or greater for all AMR genes. The BIOFIRE JI Panel provides an improvement over SOC culture, with a substantially shorter time to result for both organisms and AMR genes with excellent sensitivity/PPA and specificity/NPA, and is anticipated to provide timely and actionable diagnostic information for joint infections in a variety of clinical scenarios.

Case report: Continuous infusions of ceftazidime-avibactam and aztreonam in combination through elastomeric infusors for 12 weeks for the treatment of bone and joint infections due to metallo-ß-lactamase producing Enterobacterales.

Among carbapenem-resistant Enterobacterales, metallo-beta-lactamase producing strains represent a growing therapeutic challenge. While the association of aztreonam and ceftazidime-avibactam has been investigated in recent years for the treatment of infections involving these strains, little to no clinical data support the use of this association for the treatment of bone and joint infections. We report two cases of complex bone and joint infections involving metallo-beta-lactamase-producing Enterobacterales, successfully treated at our referral center with aztreonam and ceftazidime-avibactam for 12 weeks in continuous infusions through elastomeric infusors.

Molecular characterization and antimicrobial resistance of nasal Staphylococcus aureus in the community of Kabul.

OBJECTIVES: This study aimed to investigate the prevalence and molecular characteristics of community methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage among students at Kabul University. METHODS: Nasal swabs were collected from anterior nares of 150 healthy non-medical students at Kabul University. Antimicrobial susceptibility testing was performed on all S. aureus isolates, and all detected MRSA isolates were then confirmed by mecA/mecC polymerase chain reaction and characterized using DNA microarray. RESULTS: A total of 50 S. aureus strains were isolated from the anterior nares of the 150 participants. The prevalence of S. aureus and MRSA nasal carriage among Kabul students was 33.3% and 12.7%, respectively. Seven (36.8%) MRSA isolates and 8 (25.8%) methicillin-susceptible S. aureus (MSSA) isolates were multidrug-resistant (i.e. resistant to at least three different antimicrobials tested). All MRSA isolates (n = 19) were susceptible to linezolid, rifampicin, and fusidic acid. Seven MRSA clones, belonging to four clonal complexes (CCs), were identified. The most commonly identified clone was CC22-MRSA-IV TSST-1-positive, which accounted for 63.2% (12/19) of MRSA isolates. SCCmec typing showed that most MRSA strains harboured SCCmec type IV (94.7%). Thirteen (68.4%) MRSA isolates carried the TSST-1 and 5 (26.3%) PVL genes. CONCLUSION: Our findings revealed the relatively high prevalence of MRSA nasal carriers in the community in Kabul, with the predominance of the CC22-MRSA-IV TSST-1-positive clone and frequent multidrug resistance among these isolates.

Clinical, Bacteriological, and Genetic Characterization of Bone and Joint Infections Involving Linezolid-Resistant Staphylococcus epidermidis: a Retrospective Multicenter Study in French Reference Centers.

The choice of the best probabilistic postoperative antibiotics in bone and joint infections (BJIs) is still challenging. Since the implementation of protocolized postoperative linezolid in six French referral centers, linezolid-resistant multidrug-resistant Staphylococcus epidermidis (LR-MDRSE) strains were isolated in patients with BJI. We aimed here to describe clinical, microbiological, and molecular patterns associated with these strains. All patients with at least one intraoperative specimen positive for LR-MDRSE between 2015 and 2020 were included in this retrospective multicenter study. Clinical presentation, management, and outcome were described. LR-MDRSE strains were investigated by MIC determination for linezolid and other anti-MRSA antibiotics, characterization of genetic determinants of resistance, and phylogenetic analysis. Forty-six patients (colonization n = 10, infection n = 36) were included in five centers, 45 had prior exposure to linezolid, 33 had foreign devices. Clinical success was achieved for 26/36 patients. Incidence of LR-MDRSE increased over the study period. One hundred percent of the strains were resistant to oxazolidinones, gentamicin, clindamycin, ofloxacin, rifampicin, ceftaroline, and ceftobiprole, and susceptible to cyclins, daptomycin, and dalbavancin. Susceptibility to delafloxacin was bimodal. Molecular analysis was performed for 44 strains, and the main mutation conferring linezolid resistance was the 23S rRNA G2576T mutation. All strains belonged to the sequence type ST2 or its clonal complex, and phylogenetic analysis showed emergence of five populations corresponding geographically to the centers. We showed the emergence of new clonal populations of highly linezolid-resistant S. epidermidis in BJIs. Identifying patients at risk for LR-MDRSE acquisition and proposing alternatives to systematic postoperative linezolid use are essential. IMPORTANCE The manuscript describes the emergence of clonal linezolid-resistant strains of Staphylococcus epidermidis (LR-MDRSE) isolated from patients presenting with bone and joint infections. Incidence of LR-MDRSE increased over the study period. All strains were highly resistant to oxazolidinones, gentamicin, clindamycin, ofloxacin, rifampicin, ceftaroline, and ceftobiprole, but were susceptible to cyclins, daptomycin, and dalbavancin. Susceptibility to delafloxacin was bimodal. The main mutation conferring linezolid resistance was the 23S rRNA G2576T mutation. All strains belonged to the sequence type ST2 or its clonal complex, and phylogenetic analysis showed emergence of five populations corresponding geographically to the centers. LR-MDRSE bone and joint infections seem to be accompanied by an overall poor prognosis related to comorbidities and therapeutic issues. Identifying patients at risk for LR-MDRSE acquisition and proposing alternatives to systematic postoperative linezolid use become essential, with a preference for parenteral drugs such as lipopeptids or lipoglycopeptids.

Staphylococcus aureus nasal colonization level and intracellular reservoir: a prospective cohort study.

Staphylococcus aureus is a major pathogen in humans. The nasal vestibule is considered as the main reservoir of S. aureus. However, even though the nasal cavity may also be colonized by S. aureus, the relationships between the two sites are still unclear. We conducted a prospective study in humans to assess the S. aureus colonization profiles in the vestibule and nasal cavity, and to investigate the presence of intracellular S. aureus in the two sites. Patients undergoing ear, nose, and throat surgery were swabbed during endoscopy to determine S. aureus nasal load, genotype, and presence of intracellular S. aureus. Among per-operative samples from 90 patients, the prevalence of S. aureus carriage was 32.2% and 33.3% in the vestibule and the nasal cavity, respectively. The mean S. aureus load was 4.10 and 4.25 log10 CFU/swab for the nasal vestibule and nasal cavity, respectively (P > 0.05). Genotyping of S. aureus revealed that all nasal strains isolated from a given individual belong to the same clonal complex and spa-type. An intracellular carriage was observed in 5.6% of the patients, all of whom exhibited a S. aureus vestibule load higher than 3 log10 CFU/swab. An intracellular niche was observed in the vestibule as well as in the nasal cavity. In conclusion, the nasal cavity was also found to be a major site of S. aureus carriage in humans and should draw attention when studying host-pathogen interactions related to the risk of infection associated with colonization.

Efficacy of ceftazidime/avibactam in various combinations for the treatment of experimental osteomyelitis in rabbits caused by OXA-48-/ESBL-producing Escherichia coli.

BACKGROUND: While the treatment of ESBL-producing Enterobacterales osteomyelitis relies on carbapenems, the optimal regimen for OXA48 types remains unclear. We evaluated the efficacy of ceftazidime/avibactam in different combinations in an experimental model of OXA-48-/ESBL-producing Escherichia coli osteomyelitis. METHODS: E. coli pACYC184 is a clinical strain harbouring blaOXA-48 and blaCTX-M-15 inserts, with 'increased exposure susceptibility' to imipenem (MIC, 2 mg/L), gentamicin (MIC, 0.5 mg/L), colistin (MIC, 0.25 mg/L), ceftazidime/avibactam (MIC, 0.094 mg/L) and fosfomycin (MIC, 1 mg/L), and resistance to ceftazidime (MIC, 16 mg/L). Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu of OXA-48/ESBL E. coli. Treatment started 14 days later for 7 days in six groups: (1) control, (2) colistin 150.000 IU/kg subcutaneously (SC) q8h, (3) ceftazidime/avibactam 100/25 mg/kg SC q8h, (4) ceftazidime/avibactam + colistin, (5) ceftazidime/avibactam + fosfomycin 150 mg/kg SC q12h, (6) ceftazidime/avibactam + gentamicin 15 mg/kg intramuscularly (IM) q24h. Treatment was evaluated at Day 24 according to bone cultures. RESULTS: In vitro, time-kill curves of ceftazidime/avibactam in combination showed a synergistic effect. In vivo, compared with controls, rabbits treated with colistin alone had similar bone bacterial density (P = 0.50), whereas ceftazidime/avibactam alone or in combinations significantly decreased bone bacterial densities (P = 0.004 and P < 0.0002, respectively). Bone sterilization was achieved using ceftazidime/avibactam in combination with colistin (91%) or fosfomycin (100%) or gentamicin (100%) (P < 0.0001), whereas single therapies were not different from controls. No ceftazidime/avibactam-resistant strains emerged in rabbits treated, regardless of the combination. CONCLUSIONS: In our model of E. coli OXA-48/ESBL osteomyelitis, ceftazidime/avibactam in combination was more effective than any single therapy, whatever the companion drug used (gentamicin or colistin or fosfomycin).

Sensitivity of the PBP2a SA Culture Colony Test on shortly incubated subcultures of methicillin-resistant staphylococci from positive blood cultures.

The sensitivity of an immunochromatographic assay for detecting methicillin resistance (PBP2a SA Culture Colony Test, Alere-Abbott) on shortly incubated subcultures of staphylococci in blood cultures was evaluated. The assay is highly sensitive for the detection of methicillin-resistant Staphylococcus aureus after 4 hour-subculture but requires 6 hour-incubation for methicillin-resistant coagulase-negative staphylococci.

Disinfection of incubators in neonatal intensive care units: impact of steam pulverization on bacterial colonization.

BACKGROUND: In neonatal intensive care units (NICUs), neonates requiring medical care after birth, including very vulnerable preterm infants, are housed in incubators. Previous studies have reported that the standard chemical disinfection measures used to disinfect these incubators are insufficient to eradicate contaminating bacteria, leading to a worrying infectious risk for preterm neonates. This study aimed to evaluate the efficacy of a disinfection method based on steam pulverization to eradicate the persistent bacterial contamination in such incubators. METHODS: In a tertiary NICU, 20 incubators were monitored qualitatively for bacterial contamination at five different sites (the rubber grommet, the left door handles, the temperature adjustment button, the mattress and the scale) using a culture method at three times: before and after steam pulverization then 24 h after turning on and housing a new neonate. Clinical data of neonates housed in each incubator were retrieved from the medical records to identify potential occurrence of late onset sepsis (LOS). RESULTS: Just after steam pulverization, only two incubators were free from bacteria. Before disinfection 87% of all the samples were contaminated compared to 61% after disinfection. After 24 h, the proportion of contaminated samples reached 85%. Mattresses and scales were the most frequently contaminated incubator sites with respectively 90% and 80% positive samples after disinfection compared to 100% and 90% before disinfection. Coagulase-negative staphylococci, Enterococcus, Enterobacteria and Bacillus resisted disinfection and were identified on respectively 90%, 20%, 5% and 45% of incubators just after disinfection. Three preterm neonates developed LOS after being housed in a disinfected incubator but the bacterial species involved have not been identified in their incubator after disinfection. In two cases, the bacterium had been isolated from the mattress 24 h after housing the infected patient. CONCLUSION: Steam pulverization is not sufficient to eradicate bacterial contamination of incubators. These results highlight the urgent need for an effective disinfection method, especially for mattresses that are in constant contact with patients. In parallel, new incubator designs and mattress protections must be developed.

Evaluation of ceftolozane-tazobactam susceptibility on a French nationwide collection of Enterobacterales.

OBJECTIVES: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020. METHODS: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum ß-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison. RESULTS: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-ß-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement. CONCLUSION: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference.

Linezolid resistance: detection of the cfr(B) gene in French clinical MRSA strains.

OBJECTIVES: To describe two linezolid-resistant MRSA strains carrying the cfr(B) gene detected in the French National Reference Centre for staphylococci. METHODS: Two linezolid-resistant MRSA strains isolated from cystic fibrosis patients in two different French hospitals in 2017 and 2019 were examined to explore the mechanisms of linezolid resistance. Antimicrobial susceptibility was tested using broth microdilution and gradient strips. The genetic determinants of linezolid resistance were assessed by a multiplex PCR targeting cfr/cfr(B), optrA and poxtA genes, by amplification and sequencing of individual 23S rRNA genes and by WGS using both Illumina and Nanopore technologies. RESULTS: The two MRSA strains were resistant to linezolid but susceptible to tedizolid, and PCR-positive for cfr/cfr(B). The WGS analysis indicated that they belonged to two different STs (ST8-MRSA-IV and ST5382-MRSA-IV) and that they both harboured the cfr(B) gene on the same 9.7 kb Tn6218-like chromosomal transposon, a finding only previously reported in Enterococcus sp. and Clostridioides difficile. CONCLUSIONS: To the best of our knowledge, this is the first description of the presence of cfr(B) in staphylococci, more specifically in linezolid-resistant MRSA strains. This finding illustrates the risk of horizontal intergenus transfer of oxazolidinone resistance genes in Staphylococcus aureus and highlights the need to monitor such emergence in this species.

Efficacy of ceftazidime-avibactam in various combinations for the treatment of experimental osteomyelitis due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae.

BACKGROUND: Optimal treatment of carbapenemase-producing Enterobacterales (CPE) bone infections is poorly defined. This study evaluated the efficacy of the novel beta-lactam-beta-lactamase inhibitor-ceftazidime-avibactam (CAZ-AVI)-with different antibiotic combinations in an experimental model of CPE osteomyelitis. METHODS: KPC-99YC is a clinical strain of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae with intermediate susceptibility to meropenem (MIC 4 mg/L), gentamicin (MIC 0.25 mg/L), colistin (MIC 0.25 mg/L), fosfomycin (MIC 4 mg/L) and ceftazidime-avibactam (MIC 1 mg/L). Time-kill curves were performed at 4x MIC. Osteomyelitis was induced in rabbits by tibial injection of 2×108 CFU of KPC-99YC. Six groups started treatment 14 days later for 7 days: control, colistin, CAZ-AVI, CAZ-AVI plus gentamicin, CAZ-AVI plus colistin and CAZ-AVI plus fosfomycin. Antibiotic dosages were selected to simulate plasma concentrations obtained in humans. Treatment was evaluated according to bone cultures quantified in log10 CFU. RESULTS: In vitro, CAZ-AVI plus colistin or gentamicin were rapidly bactericidal in contrast with CAZ-AVI plus fosfomycin. In vivo, compared with controls, colistin alone (P = 0.045) and CAZ-AVI alone or in combination significantly lowered bone bacterial counts (P < 0.001). Bone sterilisation was achieved in 67% and 100% of animals with combinations of CAZ-AVI plus colistin or gentamicin (P = 0.001 and P < 0.001, respectively) whereas other treatments were no different from controls. CAZ-AVI plus gentamicin provided greater bone bacterial reduction than CAZ-AVI plus colistin (P = 0.033). No CAZ-AVI-resistant strains emerged in treated rabbits, regardless of combination. CONCLUSIONS: CAZ-AVI plus gentamicin was the best effective combination therapy. Combinations with CAZ-AVI appear to be a promising treatment of KPC-producing Klebsiella pneumoniae osteomyelitis.

Bacteriophage-based decontamination to control environmental colonization by Staphylococcus capitis in neonatal intensive care units: An in vitro proof-of-concept.

Introduction: In neonatal intensive care units (NICUs), the standard chemical-based disinfection procedures do not allow a complete eradication of pathogens from environmental surfaces. In particular, the clone Staphylococcus capitis NRCS-A, a significant pathogen in neonates, was shown to colonize neonatal incubators. The aim of this study was to evaluate the in vitro effect of a bacteriophage cocktail on NRCS-A eradication. Methods: Three bacteriophages were isolated, genetically characterized and assessed for their host range using a collection of representative clinical strains (n=31) belonging to the clone NRCS-A. The efficacy of a cocktail including these three bacteriophages to eradicate the reference strain S. capitis NRCS-A CR01 was determined in comparison or in combination with the chemical disinfectant Surfanios Premium on either dry inoculum or biofilm-embedded bacteria. The emergence of bacterial resistance against the bacteriophages alone or in cocktail was evaluated by growth kinetics. Results: The three bacteriophages belonged to two families and genera, namely Herelleviridae/Kayvirus for V1SC01 and V1SC04 and Rountreeviridae/Andhravirus for V1SC05. They were active against 17, 25 and 16 of the 31 tested strains respectively. Bacteriophage cocktails decreased the bacterial inoculum of both dry spots and biofilms, with a dose dependent effect. The sequential treatment with bacteriophages then Surfanios Premium did not show enhanced efficacy. No bacterial resistance was observed when using the bacteriophage cocktail. Discussion: This study established a proof-of-concept for the use of bacteriophages to fight against S. capitis NRCS-A. Further investigations are needed using a larger bacterial collection and in real-life conditions before being able to use such technology in NICUs.

Environmental Persistence of Staphylococcus capitis NRCS-A in Neonatal Intensive Care Units: Role of Biofilm Formation, Desiccation, and Disinfectant Tolerance.

The clone Staphylococcus capitis NRCS-A is responsible for late-onset sepsis in neonatal intensive care units (NICUs) worldwide. Over time, this clone has evolved into three subgroups that are increasingly adapted to the NICU environment. This study aimed to decipher the mechanisms involved in NRCS-A persistence in NICUs. Twenty-six S. capitis strains belonging to each of the three NRCS-A clone subgroups and two other non-NRCS-A groups from neonates (alpha clone) or from adult patients ("other strains") were compared based on growth kinetics and ability to form biofilm as well as tolerance to desiccation and to different disinfectants. S. capitis biofilm formation was enhanced in rich medium and decreased under conditions of nutrient stress for all strains. However, under conditions of nutrient stress, NRCS-A strains presented an enhanced ability to adhere and form a thin biofilm containing more viable and culturable bacteria (mean 5.7 log10 CFU) than the strains from alpha clone (mean, 1.1 log10 CFU) and the "other strains" (mean, 4.2 log10 CFU) (P < 0.0001). The biofilm is composed of bacterial aggregates with a matrix mainly composed of polysaccharides. The NRCS-A clone also showed better persistence after a 48-h desiccation. However, disinfectant tolerance was not enhanced in the NRCS-A clone in comparison with that of strains from adult patients. In conclusion, the ability to form biofilm under nutrient stress and to survive desiccation are two major advantages for clone NRCS-A that could explain its ability to persist and settle in the specific environment of NICU settings. IMPORTANCE Neonatal intensive care units (NICUs) host extremely fragile newborns, including preterm neonates. These patients are very susceptible to nosocomial infections, with coagulase-negative staphylococci being the species most frequently involved. In particular, a Staphylococcus capitis clone named NRCS-A has emerged worldwide specifically in NICUs and is responsible for severe nosocomial sepsis in preterm neonates. This clone is specifically adapted to the NICU environment and is able to colonize and maintain on NICU surfaces. The present work explored the mechanisms involved in the persistence of the NRCS-A clone in the NICU environment despite strict hygiene measures. The ability to produce biofilm under nutritional stress and to resist desiccation appear to be the two main advantages of NRCS-A in comparison with other strains. These findings are pivotal to provide clues for subsequent development of targeted methods to combat NRCS-A and to stop its dissemination.