Coagulase

In this surveillance study, we analysed blood cultures that were routinely taken from adult and paediatric patients with fever or suspicion of sepsis admitted to QECH, Blantyre, Malawi between 1998 and 2016.
The Malawi-Liverpool-Wellcome Trust Clinical Research Programme has provided routine, quality controlled, diagnostic blood culture service for febrile adult and paediatric medical patients admitted to QECH since 1998. A recommended 7–10 mL of blood were taken for culture under aseptic conditions from all adult patients admitted to the hospital with fever (axillary temperature >37·5°C) or clinical suspicion of sepsis, severe sepsis, or septic shock.²⁹ (link) Sepsis, severe sepsis, or septic shock were suspected in patients with tachycardia (≥90 beats per minute), hypotension (systolic blood pressure <90 mm Hg), tachypnoea (respiratory rate >20 per minute), or delirium. 3–10 mL of blood was taken from children with non-focal febrile illness who tested negative for malaria, who were severely ill with suspected sepsis, or who failed initial malaria treatment and remained febrile.¹⁸ (link) In this busy hospital, afebrile patients were unlikely to have blood sampled for culture unless critically ill with suspected sepsis. If patients were critically ill and sample for culture was taken, the patients were not excluded from analysis.
Since 2000, blood was inoculated into a single aerobic bottle using the automated BacT/ALERT system (bioMérieux, France)⁶ (link) before which, manual culture was used.³⁰ (link) Enterobacteriaceae and oxidase-positive Gram-negative bacilli were identified by API (BioMérieux, France), staphylococci by tube coagulase, β-haemolytic streptococci by Lancefield antigen testing, and salmonella by serotyping according to the White-Kauffmann-Le Minor scheme by the polyvalent O & H, O4, O9, Hd, Hg, Hi, Hm, and Vi antisera (Pro-Lab Diagnostics, UK). The identification of a sample of isolates as Salmonella enterica serotype Typhimurium was subsequently substantiated by whole genome sequencing and multi-locus sequence typing. Haemophilus influenzae was typed using type B antisera. Bacteria that form part of the normal skin or oral flora, including diphtheroids, bacilli, micrococci, coagulase-negative staphylococci, and α-haemolytic streptococci (other than S pneumoniae), were considered to be contaminants.³¹
Antimicrobial susceptibility tests were done by the disc diffusion method following the British Society of Antimicrobial Chemotherapy (BSAC) methods and breakpoints. Testing was in most cases limited to one plate containing six discs, and the choice of agent varied depending on the range of antimicrobials available to clinicians. Standard operating procedures are included in the appendix. Bacteria were defined as being resistant to Malawian first-line drugs (hereafter, RFL) if they were resistant to the three first-line antimicrobials commonly used in Malawi: amoxicillin, co-trimoxazole, and chloramphenicol for Gram-negative isolates; or penicillin, co-trimoxazole, and chloramphenicol for Gram-positive isolates. Isolates were considered multidrug resistant if they were resistant to three or more classes of antimicrobials to which reference strains are susceptible.³² (link) Gram-negative isolates have been screened for ESBL-producing status using a cefpodoxime disc since 2007. Before then, ESBL was inferred on the basis of resistance to ceftriaxone. Meticillin resistance in S aureus was inferred by cefoxitin resistance, which replaced oxacillin resistance testing in 2010.

Bulk farm milk samples were cultured to determine milk quality indicators such as TBC, CC, and CPS. Sample preparation was performed following the International Organization for Standardization protocol (ISO 8261:2001) [40 ]. Tenfold serial dilution of milk was performed by transferring 1 ml of milk of the previous dilution into 9 ml of 0.1% peptone water and was mixed by vortex. One milliliter of milk was discarded from the last dilution. Each sample was serially diluted up to 10^− 8 for TBC and 10^− 6 for CC and CPS.
TBC was enumerated on plate count agar (HiMedia Ltd., Mumbai, India) according to ISO 4833:2003 [41 ], while CC was counted on violet red bile agar (HiMedia Ltd., Mumbai, India) according to ISO 4832:2006 [42 ] using the pour plate method in both cases. One hundred microliters of serially diluted milk was aseptically withdrawn from each dilution using a micropipette and plated using 15–20 ml plate count agar and violet red bile agar, which were kept at 47 °C in a water bath. After thorough mixing by rotating, the plated samples were allowed to solidify and incubated aerobically at 30 °C for 48–72 hrs and 24 hrs for TBC and CC, respectively.
For enumeration of CPS (Staphylococcus aureus and other species), 100 μL of serially diluted milk sample was transferred to Baird-Parker agar supplemented with 20% egg yolk and 3.5% potassium tellurite (Oxoid Ltd., Basingstoke, England) and spread by a bent glass rod. The plates were incubated at 37 °C for 24 hrs and the positions of typical colonies were marked on the bottom of the plates. The plates were reincubated for an additional 24 hrs, and new typical colonies were marked. Two to five typical staphylococcal colonies were picked and a coagulase test was performed. Typical colonies are black or grey, shining, convex, and surrounded by a clear zone [44 ].
Two consecutive petri dishes with colony counts between 30 and 300 per plate were considered for TBC, while plates that contained 10–100 and 15–300 colonies were considered for CC and CPS, respectively. Then, the bacterial count in the respective original sample was expressed as the number of colony forming units per ml (cfu/ml) of samples according to ISO 7218:2007 [43 ].

Isolation and identification of S. aureus was performed according to ISO 6888-1:1999 [44 ] with minor modifications. Milk samples were enriched overnight in tryptone soya broth (HiMedia, India) at 37 °C to improve the recovery of injured cells (ISO 6888-3:2004) [45 ]. The swab samples were incubated in peptone water overnight at 37 °C. A loopful of culture was streaked on Baird-Parker agar supplemented with egg yolk and potassium tellurite and incubated at 37 °C for 24–48 hours. Up to five well-isolated typical colonies were picked and inoculated into nutrient broth (HiMedia, India) and incubated at 37 °C for 24 hrs. Then, the broth culture was plated onto nutrient agar plates and incubated at 37 °C for 24 hrs for purification and further identification. The presumptive S. aureus colonies were further identified based on Gram staining, mannitol fermentation (HiMedia, India), catalase activity, and coagulase activity using freeze-dried rabbit plasma (Santa Fe Drive, Lenexa, USA).