Bactec peds plus f culture vial

Upon clinical suspicion of systemic infection, at least two blood samples were injected into BD BACTEC™ Peds Plus TM/F culture vials (enriched Soybean Casein Digest Broth with CO₂) and upon arrival at the lab immediately processed in the BACTEC FX (Becton Dickinson, Heidelberg, Germany) blood culture system. Blood cultures were incubated for five days. On positivity, Gram-staining and sub-cultivation on agar plates were performed according to the standard techniques [21 ]. Positive samples were cultivated on Columbia Blood Broth, chocolate, MacConkey and Schaedler Anaerobic Agar culture media (all Becton Dickinson, Heidelberg, Germany) and incubated for 24 h at 37 °C in aerobic and 48 h in anaerobic conditions.
Species identification of positive cultures was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonik, Bremen, Germany) using the reference Biotyper library v4.1 (Bruker Daltonik, Bremen, Germany). Antimicrobial susceptibility testing was performed according to NCCLS/CLSI guidelines until 2011 [22 ]. In 2011, Austrian microbiological laboratories switched their methodology to EUCAST (breakpoint tables for interpretation of MICs and zone diameters—2011–2021, Versions 1.3 to 11.0). Strains were classified as susceptible or resistant according to the breakpoints applied in the year of their isolation.

Before treatment, routine sepsis workup was performed in all neonates who were suspected of clinical sepsis and the laboratory parameters included in the EMA criteria were evaluated in all patients (complete blood count, C-reactive protein, blood gases, blood glucose, and immature/total neutrophil ratio with peripheral smear). At least 1 mL blood was taken for blood culture (BD BACTEC Peds Plus/F Culture vials, Becton, Dickinson and Company, USA) and the samples were processed according the Clinical and Laboratory Standards Institute (CLSI) guideline [6 ]. According to the attending neonatologists’ decision, other body fluid samples (i.e. urine, cerebrospinal fluid, tracheal aspirate) were evaluated and additional studies (i.e. metabolic tests, radiological imaging) were performed for differential diagnosis.

Neonatal sepsis could be caused by one or more systemic infections such as septicemia, urinary tract infections, pneumonia, meningitis, arthritis, or osteomyelitis; nevertheless, neonates with positive blood culture results were considered confirmed sepsis cases [9 (link)].
1-2 ml of peripheral blood samples were drawn from all neonates, those having a presumptive diagnosis of sepsis. Aseptically, the samples were injected into BD-BACTEC Peds Plus/F culture vials (Becton Dickinson, UK) and incubated in an automated BD BACTEC FX40 (Becton Dickinson, UK) culture system. Upon an indication of culture-positive, a small drop (approximately a loopful volume) of blood was aspirated and inoculated on 5% sheep blood agar, MacConkey agar, and chocolate agar. Further, isolation and identification of the isolates were done by standard microbiological techniques—biotyping (colony morphology, staining reaction, and biochemical characteristics) and serotyping using specific antisera(Denka Seiken Co. Ltd., Tokyo, Japan). The antimicrobial susceptibility testing (AST) was performed by the disk diffusion method [modified Kirby-Bauer method] on Mueller Hinton agar (Hi-Media, India) in compliance with standard procedures recommended by the Clinical and Laboratory Standards Institute (CLSI), Wayne, PA, USA [10 ].

One to two ml of peripheral blood samples were drawn from the neonates clinically suspected of sepsis and before the administration of antimicrobials. The samples were aseptically injected into BD-BACTEC Peds Plus/F culture vials (Becton Dickinson, UK) and incubated in an automated BD BACTEC FX40 (Becton Dickinson, UK) culture system. Upon being flagged as culture positive, small volume of blood was aseptically aspirated with sterile syringe and inoculated on 5% sheep blood agar, MacConkey agar and chocolate agar. Bacterial identification was conducted by standard microbiological methods and antimicrobial susceptibility testing (AST) was performed using modified Kirby-Bauer disc diffusion method [9 (link)],with inhibition zone sizes interpreted according to Clinical Laboratory Standards Institute (CLSI) 2017 breakpoint guidelines [10 ]. Gram-negative bacilli (GNB) that were resistant (zone of inhibition ≤22 mm) or intermediate (zone of inhibition 23-25 mm) to cefotaxime (30 μg) were suspected as producing extended-spectrum beta-lactamases (ESBL).

Blood was taken with aseptic technique, directly inoculated into aerobic (BD BACTEC Plus Aerobic/F) and anaerobic culture vials (BD BACTEC Lytic/10 Anaerobic/F), or peds culture vials (BD BACTEC Peds Plus/F culture vials) (Becton Dickinson & Co, BD, Shanghai, China). Each adult patient needed an Aerobic and an anaerobic culture vials, the Peds vials were used only for pediatric patients. All blood culture bottles were loaded onto the BD BACTEC FX instrument (Becton Dickinson, Franklin Lakes, NJ, USA).

The study was conducted at seven hospitals in Federal Capital Territory (FCT) and three in Kano Nigeria as previously described from Sept 2008—Dec 2016 [1 (link), 23 (link)]. Children aged less than five years were enrolled at the different sites, blood specimens of 1–3 ml were collected using a vacutainer set, after aseptically cleansing the skin with alcohol swab and povidone-iodine, the specimen was collected directly into an aerobic blood culture bottle (BD BactecPeds Plus/F culture vials; Becton Dickinson, Ireland), and incubated in an automated Bactec^® 9050 machine. All positive bottles were sub cultured onto MacConkey, Sheep blood and chocolate agar plates at 36°C for 24 h.
A total number of 887 culture-positive Enterobacteriaceae were obtained. Of the 887 isolates, 474 salmonella species including Typhi which have been reported by Obaro et al. 2015 were excluded [23 (link)], therefore 413 including Escherichia coli, Klebsiella species, Enterobacter species, Serratia marcescens, Pantoea species, Salmonella Typhi and Citrobacter species from September 2008 to December 2016 were included. The isolates were stored in 10% skim milk glycerol at -80°C.