This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages: (1) phage infection of target bacterium; (2) destruction of exogenous phage; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml-1 in a time of 4 h and 600 bacteria m-1 for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.
Aims: To develop and evaluate an antimicrobial supplement for use with phage‐based tests for rapid detection of drug resistance of tuberculosis (TB). Methods and Results: An antimicrobial formulation containing nystatin, oxacillin and aztreonam (NOA) (final concentrations of 50 000 IU l−1, 2 mg l−1, and 30 mg l−1 respectively) was developed. This formulation was tested for its influence on detection of a number of Mycobacterium tuberculosis (MTB) strains using the phage amplification (FASTPlaque) assay. Addition of the supplement did not lead to significant reduction in assay sensitivity. Antimicrobial efficacy was assessed with a range of Gram‐positive and ‐negative organisms. The NOA supplement had a broad antimicrobial effect. The supplement was tested for its effect on growth of MTB culture, and on determination of rifampicin resistance using the phage‐based methodology (FASTPlaque–Response). NOA did not significantly affect the growth of a range of rifampicin susceptible and resistant MTB strains, nor did it have an adverse effect on the number of interpretable results, nor the ability to discriminate between rifampicin susceptibility and resistance. Conclusion, Significance and Impact of Study: Use of NOA antimicrobial supplement with rapid phage‐based tests for TB will increase the proportion of interpretable results obtained, and enable their wider implementation in disease‐endemic countries by improved control of specimen‐related contamination.
BackgroundAntimicrobial resistance is a pressing global health issue. Data are lacking in detailing the presence and burden of antimicrobial resistance in low and middle-income countries. What is currently available is quarantined to large, urban centers away from the rural facilities. MethodsThis was a retrospective descriptive study performed at Kijabe Hospital, a rural 350-bed teaching hospital, from February 2016 to September 2020. Cultures from blood, urine, and cerebrospinal fluid were included from all pediatric and adult patients. Data was analyzed and an antibiogram was created using WHONET software. ResultsFrom January 2016 to September 2020 a total of 3275 distinct isolates were identified, including 1654 positive blood cultures, 1288 positive urine cultures, and 91 positive cerebrospinal fluid cultures. Aggregate gram negative susceptibility to third generation cephalosporins was approximately 41%, with 67% of isolates susceptible to piperacillin-tazobactam, and 93% of isolates susceptible to meropenem. The most frequently identified organism was coagulase-negative Staphylococcus (1534, 47%), followed by Escherichia coli (721, 22%), Klebsiella species (482, 15%), and Staphylococcus aureus (110, 3.4%). The most common multidrug resistant organism was Escherichia coli (664, 20%), followed by Klebsiella species (461, 14%). Acinetobacter baumannii was found to be only 57% sensitive to meropenem. Staphylococcus aureus was 91% sensitive to cloxacillin.ConclusionThe high rates of antimicrobial resistance found in this rural referral center were similar to the large urban settings in sub-Saharan Africa. This along with the discovery of multidrug resistant gram negative organisms are of great concern. The need for continued surveillance, antimicrobial stewardship, and implementation of quality improvement initiatives is imperative to attempt to curb this burgeoning global problem.
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