Methicillin Resistance

The strain collection consisted of MRSA from various clinical sources (e.g., blood cultures and wound infections) and included surveillance cultures from patients and staff. Of all clinical S. aureus isolates, 6.4% exhibited methicillin resistance in 2003. For species identification, every strain was tested with API ID 32 Staph (bioMérieux, Marci l'Etoile, France) and for the presence of free coagulase. The presence of the mecA gene responsible for methicillin resistance was confirmed using PCR [18 (link)]. The sequence of the short sequence repeat region of the spa gene encoding the S. aureus protein A was determined in 557 strains [14 (link)]. The primers spa-1113f (5′- TAA AGA CGA TCC TTC GGT GAG C −3′) and spa-1514r (5′- CAG CAG TAG TGC CGT TTG CT −3′) were used for spa amplification and Taq Cycle sequencing. DNA sequences were obtained with an ABI Prism 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, California, United States) and analyzed with the Ridom StaphType software version 1.5 beta (Ridom GmbH, Würzburg, Germany) incorporating the newly added automated early warning system (“clonal alerts”) for MRSA cluster detection [14 (link)]. Typability, discriminatory index, and the 95% confidence interval (CI) of the discriminatory index were calculated using the procedures published previously [19 (link),20 (link)].

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.

The de novo-assembled genomes were interrogated with BLAST+ (v 2.2.28+) blastn and tblastn (9 (link)) to identify nucleotide sequences matching genes from the panel and their matching protein sequences, respectively. The parameters for the two programs were as follows: for blastn, word size of 17, gap opening penalty of 5, and gap extension penalty of 2; for tblastn, word size of 3, gap opening penalty of 11, and gap extension penalty of 1. The E-value cutoff was set at 0.001. Relative coverage was defined as the product of the proportion of reference allele matched and the sequence identity of the match. For the initial algorithm (v1.0), a relative coverage threshold of >80% was chosen to define gene presence with a high degree of similarity to the reference, based on pilot data (for example, 95% relative coverage may be 95% of the gene length with 100% identity or 100% of the gene length with 95% identity). For housekeeping genes, where resistance is conferred by one or more point mutations, differences between the tblastn result and the query protein sequence were compared to the sequence of the wild-type protein and compared against the catalogue of known antimicrobial resistance-encoding mutations compiled above. Changes in protein sequence (at the same or different codons) which were not previously reported as conferring resistance were counted as susceptible.
To determine the diversity of the isolates tested, in silico prediction of multilocus sequence type (MLST) was also performed using BLAST+. The S. aureus MLST alleles were extracted from assemblies based on sequence similarity to allele 1 for each locus, and the online MLST database (http://saureus.mlst.net/) was used to predict the ST.
The initial development of the algorithm was not done in a blind manner, using 501 clinical S. aureus isolates which had been sequenced and phenotyped previously and whose WGS and resistance data were available (“derivation set”). To ensure a representative range of sequence types, isolates were identified from bacteremia and carriage collections held at the Oxford Radcliffe Hospitals NHS Trust and Brighton and Sussex University Hospitals NHS Trust, spanning a period of 13 years (Table 3) (10 (link)). The collection included 159 MRSA isolates (32%). All isolates had been tested at each site by the routine clinical laboratories for resistance to a standard first-line panel of antimicrobial agents (penicillin, methicillin, erythromycin, vancomycin, ciprofloxacin, tetracycline, gentamicin, fusidic acid, and rifampin at both sites; mupirocin and clindamycin for Brighton isolates only; trimethoprim for Oxford isolates only). In the Brighton clinical laboratory, susceptibility testing was performed using the Vitek automated system (bioMérieux, Basingstoke, United Kingdom), and in the Oxford clinical laboratory, isolates were phenotyped by disc diffusion (11 (link)). The susceptibility testing results were retrieved electronically from laboratory databases. Methicillin resistance was tested using cefoxitin (Brighton) or oxacillin (Oxford).

Microorganisms used in this study included L. enzymogenes ATCC 29487, methicillin-sensitive S. aureus (MSSA) (ATCC 25923), methicillin-resistance S. aureus (MRSA) (ATCC 33591) and E. coli O157:H7. Culture media used included brain heart infusion (BHI) agar and broth obtained from Sigma- Aldrich (India) and nutrient agar (NA) obtained from Scharlau (European Union). Sterile syringe filters (0.22 μm) were purchased from Millipore (Amman, Jordan). Antibiotics (cefixime, gentamicin and levofloxacin) were kindly donated by JOSWE^® Medical and Dar Al Dawa^® pharmaceutical company, Amman-Jordan.
L. enzymogenes bacterial pellets were rehydrated aseptically using 5 mL of 10% strength tryptone soy broth (TSB), incubated at 28°C and shaken at 200 rpm for 3 days in a shaker incubator (MS, Taiwan) [44 (link)]. Then, L. enzymogenes culture (200 μL) was seeded over 10% strength of tryptone soy agar (TSA) plate and incubated at 28°C for 3 days. L. enzymogenes pure broth culture (PBC) was prepared by transferring a full loop from the stock plate to a 50 mL sterile falcon containing 10% TSB, followed by incubation at 28°C for 3 days. E. coli O157:H7 was cultured in nutrient broth (NB) (Biolab); MSSA and MRSA in brain heart infusion (BHI) broth (Biolab).

S. aureus samples were obtained from The Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. A total of 13 samples were tested for the methicillin resistance using disk diffusion (24 (link)), thus determining their methicillin resistance before photoacoustic testing. Streaks of each S. aureus strain were grown on mannitol salt agar plates. Single colonies from each streak plate were used to regrow strains in mannitol salt broth for 2 h in a shaking water bath at 36.5 °C. This period ensured cells were growing and entering exponential growth phase. Oxacillin was added at a final concentration of 1 μg/ mlto half of each culture and grown for an additional 2 h (25 (link)). We cultured on agar plates solely for comparison to plate reader results. The photoacoustic method, in clinical implementation, will test samples directly taken from patients without the culture phase.
Before processing through PAFC system, 100 μLfrom each culture was removed and used for growth analysis in an H1 plate reader (Biotek, Winooski, Vermont). This procedure was done to verify the photoacoustic method and were not integral to photoacoustic testing. Growth curves were made for each culture by taking the optical density of each treated and untreated culture every minute over a 16 hour period. We determined that two hours of antibiotic treatment was sufficient to determine differential growth rates from prior experimentation. Prior to performing photoacoustic testing, treated and untreated samples were incubated side-by-side for two hours. Photoacoustic testing of treated and untreated samples for each isolate were alternated, so that both samples were tested within twenty minutes to allow for similar growth times. Thus, total bacteria number could be compared.

This prospective study was conducted in the Department of Microbiology over a period of 9 months from April 2021 to December 2021 after obtaining clearance from the Ethical Committee. Fifty non-duplicate isolates of Methicillin Resistant S. aureus strains isolated from various clinical samples were included in the study. Isolates which showed gram positive cocci in clusters on grams stain and gave positive results on catalase, slide and tube coagulase where identified as S. aureus.
Screening for methicillin resistance was done by modified Kirby Bauer disc diffusion method using cefoxitin (30 µg) discs. A zone size of ≥22 mm was interpreted as methicillin sensitive and ≤21 mm was interpreted as methicillin resistant as per Clinical and Laboratory Standards Institute (CLSI) guidelines (23 ). S. aureus American Type Culture Collection (ATCC) strain 25923 and S. aureus ATCC strain 43300 were used as susceptibility and resistance controls respectively. Isolates which were Methicillin sensitive were excluded from the study. Antimicrobial susceptibility of the isolates was done by Kirby Bauers disc diffusion
Testing for ceftaroline susceptibility was done by E- test strip method. The ceftaroline E-test strips (0.002–32 µg/mL) was obtained from Biomerieux, France. The E- test strips were placed on the lawn culture of the organism and the plates were incubated at 37°C for 18–24 hours. MIC's were read where the ellipse intersects the MIC (Minimum Inhibitory Concentration) scale. Since E-test strip has continuous gradient, MIC values “in-between” two-fold dilutions can be obtained. These values were rounded up to next two-fold dilution before categorisation. MICs were interpreted according to EUCAST version 11.0 and CLSI 2021.
EUCAST version 11.0 for Ceftaroline (In pneumonia) - ≤1- Susceptible, > 1- Resistant
EUCAST version 11.0 for Ceftaroline (For conditions other than pneumonia)-≤1- Susceptible, >2- Resistant
CLSI 2021- ≤1- Susceptible, 2–4(Susceptible Dose Dependent), ≥8- Resistant.