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.

Targeted proteomics links virulence factor expression with clinical severity in staphylococcal pneumonia.

Introduction: The bacterial pathogen Staphylococcus aureus harbors numerous virulence factors that impact infection severity. Beyond virulence gene presence or absence, the expression level of virulence proteins is known to vary across S. aureus lineages and isolates. However, the impact of expression level on severity is poorly understood due to the lack of high-throughput quantification methods of virulence proteins. Methods: We present a targeted proteomic approach able to monitor 42 staphylococcal proteins in a single experiment. Using this approach, we compared the quantitative virulomes of 136 S. aureus isolates from a nationwide cohort of French patients with severe community-acquired staphylococcal pneumonia, all requiring intensive care. We used multivariable regression models adjusted for patient baseline health (Charlson comorbidity score) to identify the virulence factors whose in vitro expression level predicted pneumonia severity markers, namely leukopenia and hemoptysis, as well as patient survival. Results: We found that leukopenia was predicted by higher expression of HlgB, Nuc, and Tsst-1 and lower expression of BlaI and HlgC, while hemoptysis was predicted by higher expression of BlaZ and HlgB and lower expression of HlgC. Strikingly, mortality was independently predicted in a dose-dependent fashion by a single phage-encoded virulence factor, the Panton-Valentine leucocidin (PVL), both in logistic (OR 1.28; 95%CI[1.02;1.60]) and survival (HR 1.15; 95%CI[1.02;1.30]) regression models. Discussion: These findings demonstrate that the in vitro expression level of virulence factors can be correlated with infection severity using targeted proteomics, a method that may be adapted to other bacterial pathogens.

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.

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.

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.

Phage Therapy against Staphylococcus aureus: Selection and Optimization of Production Protocols of Novel Broad-Spectrum Silviavirus Phages.

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control.

A fusidic acid-resistant (PVL+) clone is associated with the increase in methicillin-resistant Staphylococcus aureus in New Caledonia.

OBJECTIVES: Since 2014, Staphylococcus aureus methicillin resistance has been rapidly increasing in New Caledonia and is associated with potential serious clinical repercussions. In the present study, we investigated the epidemiology of methicillin-resistant S. aureus (MRSA) in New Caledonia and the possible emergence of a particular clonal strain. METHODS: An overview of the distribution of MRSA in New Caledonia in 2019 is presented. We collected and analysed 171 clinical MRSA isolates from New Caledonia medical laboratories during August and September 2019. Among this collection, 49 representative isolates were analyzed by the French National Reference Center for Staphylococci using the StaphyType DNA microarray, allowing genetic characterization of the isolates. RESULTS: Among the 1144 S. aureus isolated over the year 2019, 442 isolates (39%) were resistant to methicillin, and 62% of these isolates were resistant to fusidic acid (FA). During the inclusion period, FA resistance rate was similar (60%). Genetic characterization evidenced CC6 as the predominant clonal complex (70%) with 26 isolates (53%) identified as CC6-MRSA-[IV+fus] (PVL+). CONCLUSIONS: These findings demonstrated a low diversity of MRSA in New Caledonia, with the dominance of a clonal complex not reported previously. The frequent fusidic acid (FA) resistance in MRSA was associated with a high prevalence of fusC gene, suggesting that FA misuse contributed to driving the selection of this clone. Our findings suggest the recommendation to stop the topical use of FA to control the emergence of this severe MRSA clone and decrease the rate of MRSA in New Caledonia.

Persistence of a multidrug-resistant worldwide-disseminated methicillin-resistant Staphylococcus epidermidis clone harbouring the cfr linezolid resistance gene in a French hospital with evidence of interspecies transfer to several Staphylococcus aureus lineages.

BACKGROUND: Resistance to linezolid has become a worldwide concern since it is one of the last-resort antibiotics to treat multidrug-resistant staphylococcal and enterococcal infections. OBJECTIVES: We investigated staphylococcal infections caused by 16 cfr-positive linezolid-resistant Staphylococcus epidermidis and Staphylococcus aureus isolates in a French university hospital from 2015 to 2018. METHODS: Antimicrobial susceptibility of isolates was tested by broth microdilution and gradient strips. Genetic determinants of linezolid resistance (including cfr gene and 23S rRNA mutations) were assessed by PCR and WGS; the latter was also used to characterize the cfr-carrying plasmids in S. epidermidis and S. aureus, and to explore the clonal relationship of isolates. RESULTS: All linezolid-resistant staphylococcal isolates harboured the same cfr-carrying plasmid, sharing 99% identity with the previously described pSA737. The three S. aureus isolates belonged to different STs (ST8, ST72, ST2416); the 13 methicillin-resistant S. epidermidis (MRSE) belonged to ST2 and harboured both cfr and mutations in genes encoding 23S rRNA and ribosomal proteins. Phylogenetic analysis grouped the MRSE isolates into two clusters, one of which (n = 12 isolates) belonged to the recently reported multidrug-resistant worldwide-disseminated S. epidermidis lineages. CONCLUSIONS: The results presented herein highlight the persistence and efficient spread of a cfr-carrying plasmid in a hospital related both to the dissemination of a multidrug-resistant S. epidermidis clone and the in vivo interspecies transfer of cfr between S. epidermidis and S. aureus. The emergence of linezolid-resistant strains should be closely monitored, and the mechanisms involved systematically explored in order to limit the spread of plasmid-mediated resistance.

Sub-Inhibitory Concentrations of Oxacillin, but Not Clindamycin, Linezolid, or Tigecycline, Decrease Staphylococcal Phenol-Soluble Modulin Expression in Community-Acquired Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus (SA) is a major human pathogen producing virulence factors, such as Panton-Valentine-leucocidin (PVL), alpha-hemolysin (Hla), and phenol-soluble-modulins alpha (PSMα), including delta-hemolysin (Hld). Unlike oxacillin, clindamycin and linezolid subinhibitory concentrations (sub-MIC) display an anti-toxin effect on PVL and Hla expression. Few studies have investigated PSMα and Hld expression modulation by antibiotics. Herein, we assessed the effect of antibiotic sub-MIC on PSMα1 and Hld expression for 4 community-acquired methicillin-resistant SA (CA-MRSA), 2 strains belonging to USASA300 and 2 strains belonging to ST80 European clone. SA were grown under oxacillin, clindamycin, linezolid, or tigecycline. After incubation, culture pellets were used for the determination of psmα1, pmtB, pmtR mRNA, and RNAIII levels by relative quantitative RT-PCR. PSMα1 and Hld expressions were measured in supernatant using high-performance-liquid-chromatography coupled to mass-spectrometry (HPLC-MS). Oxacillin sub-MIC reduced PSMα1 and Hld production, partially related to mRNA variations. For other antibiotics, effects on toxin expression were strain or clone dependent. Antibiotic effect on mRNA did not always reflect protein expression modulation. Variations of pmtB, pmtR mRNA, and RNAIII levels were insufficient to explain toxin expression modulation. Altogether, these data indicate that PSMα and Hld expressions are modulated by antibiotics (potential anti-toxin effect of oxacillin) differently compared to PVL and Hla. IMPORTANCE Staphylococcal toxins play an important role in the physiopathology of staphylococcal infections. Subinhibitory concentrations (sub-MIC) of antibiotics modulate in vitro toxins expression in S. aureus: clindamycin (CLI) and linezolid (LIN) display an anti-toxin effect on Panton-Valentine leucocidin and alpha-hemolysin production, while oxacillin (OXA) has an inducing effect. Few studies have focused on the modulation of phenol-soluble modulins alpha (PSMα) including delta-hemolysin expression by sub-MIC antibiotics. The aim of the present study was to investigate the effects of sub-MIC antibiotics on the expression of PSMα toxins for 4 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolates. The data presented herein confirm that OXA sub-MICs constantly inhibit PSMα production for CA-MRSA. Certain strains of S. aureus are highly sensitive to sub-MICs of protein synthesis inhibitory agents, resulting in an important increase of mRNA levels to overcome the intrinsic ribosome blockage ability of these antibiotics, eventually translating in increased expression of toxins.

Heterogeneous vancomycin resistance in Staphylococcus aureus does not predict development of vancomycin resistance upon vancomycin pressure.

BACKGROUND: It is unclear whether Staphylococcus aureus with heterogeneous intermediate vancomycin resistance (hVISA) can develop vancomycin resistance faster than vancomycin-susceptible S. aureus (VSSA) strains. METHODS: We compared the kinetics of vancomycin MIC increase for 15 days of sustained in vitro vancomycin exposure for clinical hVISA (n = 12) and VSSA (n = 24) isolates, as well as for reference strains Mu3 (hVISA) and ATCC 29213 (VSSA). Clinical isolates were categorized as hVISA using the population analysis profile method. MICs were monitored for 15 days and the rate of MIC increase under exposure, for each strain, was evaluated in a linear regression model relative to time. RESULTS: All isolates acquired vancomycin resistance upon exposure. Vancomycin MICs increased faster for VSSA compared with hVISA isolates (P < 0.01). CONCLUSIONS: The hVISA phenotype does not correspond to an enhanced adaptation potential to in vitro vancomycin pressure.

Menstrual Toxic Shock Syndrome: A French Nationwide Multicenter Retrospective Study.

BACKGROUND: Studies describing the clinical features and short-term prognosis of patients admitted to the intensive care unit (ICU) for menstrual toxic shock syndrome (m-TSS) are lacking. METHODS: This was a multicenter retrospective cohort study of patients with a clinical diagnosis of m-TSS admitted between 1 January 2005 and 31 December 2020 in 43 French pediatric (n = 7) or adult (n = 36) ICUs. The aim of the study was to describe the clinical features and short-term prognosis, as well as to assess the 2011 Centers for Disease and Control (CDC) diagnostic criteria, in critically ill patients with m-TSS. RESULTS: In total, 102 patients with m-TSS (median age, 18 years; interquartile range, 16-24 years) were admitted to 1 of the participating ICUs. All blood cultures (n = 102) were sterile. Methicillin-sensitive Staphylococcus aureus grew from 92 of 96 vaginal samples. Screening for superantigenic toxin gene sequences was performed for 76 of the 92 vaginal samples positive for S. aureus (83%), and toxic shock syndrome toxin 1 was isolated from 66 strains (87%). At ICU admission, no patient met the 2011 CDC criteria for confirmed m-TSS, and only 53 (52%) fulfilled the criteria for probable m-TSS. Eighty-one patients (79%) were treated with antitoxin antibiotic therapy, and 8 (8%) received intravenous immunoglobulins. Eighty-six (84%) patients required vasopressors, and 21 (21%) tracheal intubation. No patient required limb amputation or died in the ICU. CONCLUSIONS: In this large multicenter series of patients included in ICUs for m-TSS, none died or required limb amputation. The CDC criteria should not be used for the clinical diagnosis of m-TSS at ICU admission.

Adaptation of Staphylococcus aureus in a Medium Mimicking a Diabetic Foot Environment.

Staphylococcus aureus is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical S. aureus strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by Caenorhabditis elegans model (to evaluate virulence); by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes); and by Biofilm Ring test® (to assess the biofilm formation). After 16 weeks, the four S. aureus had adapted their metabolism, with the development of small colony variants and the loss of ß-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of S. aureus to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of S. aureus in chronic wounds.

Prognostic factors of severe community-acquired staphylococcal pneumonia in France.

PURPOSE: Staphylococcus aureus causes severe forms of community-acquired pneumonia (CAP), namely staphylococcal pleuropneumonia in young children and staphylococcal necrotising pneumonia in older patients. Methicillin resistance and the Panton-Valentine leukocidin (PVL) toxin, as well as less specific factors, have been associated with poor outcome in severe CAP, but their roles are unclear. METHODS: A prospective multicentre cohort study of severe staphylococcal CAP was conducted in 77 paediatric and adult intensive care units in France between January 2011 and December 2016. After age-clustering, risk factors for mortality, including pre-existing conditions, clinical presentation, laboratory features, strain genetic lineage, PVL, other virulence factors and methicillin resistance were assessed using univariate and multivariable Cox and LASSO (least absolute shrinkage and selection operator) regressions. RESULTS: Out of 163 included patients, aged 1 month to 87 years, 85 (52.1%) had PVL-positive CAP; there were 20 (12.3%) patients aged <3 years (hereafter "toddlers"), among whom 19 (95%) had PVL-positive CAP. The features of PVL-positive CAP in toddlers matched with the historical description of staphylococcal pleuropneumonia, with a lower mortality (three (15%) out of 19) compared to PVL-positive CAP in older patients (31 (47%) out of 66). Mortality in older patients was predicted by PVL-positivity (hazard ratio (HR) 1.81, 95% CI 1.03-3.17) and methicillin resistance (HR 2.37, 95% CI 1.29-4.34) independently from S. aureus lineages and the presence of other determinants of virulence. CONCLUSION: PVL was associated with staphylococcal pleuropneumonia in toddlers and was a risk factor for mortality in older patients with severe CAP, independently of methicillin resistance, S. aureus genetic background and other virulence factors.

Persistent microbial contamination of incubators despite disinfection.

BACKGROUND: In neonatal intensive care units (NICUs), hygiene and disinfection measures are pivotal to protect neonates from nosocomial infections. This study aimed to evaluate the efficacy of the classical incubators disinfection procedure and to follow-up neonates housed in the incubators for the development of late-onset sepsis (LOS). METHODS: In a tertiary NICU, 20 incubators were monitored for bacterial contamination at three times: before disinfection, after disinfection, and 24 h after turning on and housing a new neonate. Clinical data of neonates housed in these incubators were retrieved from the medical records. RESULTS: All 20 incubators were contaminated at the 3 times of the study, mainly on mattresses and balances. Coagulase-negative Staphylococci, Enterococcus, and Bacillus-resisted disinfection while enterobacteria and Staphylococcus aureus were eradicated. After 24 h, the bacterial colonisation was similar to the one observed before disinfection. The bacteria isolated on incubators were also found on the caregivers' hands. During the study, two preterm neonates developed a LOS involving a bacterial species that has been previously isolated in their incubator. CONCLUSION: Pathogenic contaminants persist on incubators despite disinfection and represent a risk for subsequent infection in preterm neonates. Improvements are needed concerning both the disinfection process and incubator design. IMPACT: Procedures of disinfection that are usually recommended in NICUs do not allow for totally eradicating bacteria from incubators. Preterm neonates are housed in incubators colonised with potentially pathogenic bacteria. The control of nosocomial infections in NICUs requires further researches concerning mechanisms of bacterial persistence and ways to fight against environmental colonisation.

Antibiotic resistance profile and molecular characterization of Staphylococcus aureus strains isolated in hospitals in Kabul, Afghanistan.

The aim of this study was to investigate the molecular features and the antibiotic resistance profile of 98 clinical Staphylococcus aureus isolates collected during 6 months in two hospitals of Kabul, Afghanistan. For all isolates, antimicrobial resistance patterns were determined by the disc diffusion method (including methicillin resistance which was detected using cefoxitin). The presence of the mecA/mecC genes was detected by PCR. Strains were then extensively characterized using microarray analysis. Of the 98 S. aureus isolates, methicillin-resistant S. aureus (MRSA) prevalence was high at 66.3%. Antibiotic susceptibility testing also revealed a high resistance rate to penicillin (100%), erythromycin (66.3%), ciprofloxacin (55.1%), and cotrimoxazole (40.8%). Resistance to tobramycin was detected in 25.5%, to gentamicin in 16.3%, to chloramphenicol in 34.7%, and to doxycycline in 23.5% of the isolates. All the MRSA isolates were mecA-positive and none of them harbored mecC. Isolates were grouped into twelve clonal complexes and twenty-seven distinct clones. The most frequently detected clones were the Southwest Pacific clone (CC30-MRSA-IV PVL+) (21/65 MRSA, 32.3%), the CC22-MRSA-IV TSST-1+ clone (11/65 MRSA, 16.9%), and the Bengal Bay clone (ST772-MRSA-V PVL+) (11/65 MRSA, 16.9%). The PVL genes were found in 59.2% (46/65 MRSA and 12/33 methicillin-susceptible S. aureus, MSSA) and tst1 gene in 16.3% of isolates. This molecular study highlights the high prevalence of MRSA and the large genetic diversity of the S. aureus isolates circulating and detected in two hospitals of Kabul, with the presence of multiple virulence and antibiotic resistance genes.

Performance of the Hologic Panther Fusion® MRSA Assay for the nasal screening of methicillin-sensitive and methicillin-resistant Staphylococcus aureus carriage.

Staphylococcus aureus (SA) nasal carriage screening is usually based on either culture or molecular biology. The aim of the study was to evaluate the performance of the Panther Fusion® MRSA Assay (PF) that proposes a complete automation of the molecular screening for MSSA and MRSA carriage. Four hundred thirty-four nasal samples collected on ESwab™ were screened using PF. Results were compared with standard culture on BBL™ CHROMagar™ Staph aureus and chromID® MRSA agar. Discordant results were analyzed with additional techniques: Xpert SA Nasal Complete on GeneXpert (GX), culture on selective agar after 24 h in broth enrichment, and, if necessary, characterization of mec gene and SCCmec cassette using DNA microarray. The PF presented an overall agreement of 97.5% for SA detection and 97.9% for MRSA detection. Furthermore, 7.1% (31/434) of the samples were SA-negative in primary culture but SA-positive using PF and GX, confirming the greater sensitivity of molecular tests compared with culture. Of note, 4 out of 30 MRSA-positive samples were not detected due to an atypical SCCmec cassette, while 2 samples were falsely detected as MRSA due to co-colonization with a MSSA drop-out strain and a methicillin-resistant coagulase-negative staphylococcal strain. Considering all results, the PF instrument appears as a reliable and rapid (< 3 h) package for MSSA/MRSA nasal screening. This technology using random access capability and direct sampling of the primary container is innovative and corresponds therefore to a new step in complete molecular biology automation in bacteriology.

Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus.

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.