Multidrug-resistant Staphylococcal Infections Research Articles

Molecular detection of multidrug and methicillin resistance in Staphylococcus aureus isolated from wild pigeons (Columba livia) in South Africa

Staphylococcus aureus is an important human and veterinary pathogen. The present study aimed to determine the prevalence of antibiotic resistance among S. aureus isolated from samples obtained from free-flying wild pigeons and houseflies from different locations surrounding a local hospital in the Greater Durban area in KwaZulu-Natal Province, South Africa. Environmental fecal samples were obtained from wild pigeons that inhabits the grounds of a local public hospital located on the South Beach area, Durban, South Africa. Housefly samples were collected from three different locations (Kenneth Stainbank Nature Reserve, Montclair/Clairwood, and Glenwood/Berea) in the greater Durban area, all within a close proximity to the hospital. Following enrichment, identification, and antimicrobial resistance profiling, S. aureus isolates were subjected to DNA extraction using the boiling method. It was found that 57 out of 252 samples (22.62%) were positive for S. aureus. The Kirby-Bauer disk diffusion method of antibiotic susceptibility testing was performed and revealed that antibiotic resistance rates to penicillin and rifampicin were the most common, with both returning 48 (84.2%) out of the 57 S. aureus isolates being resistant to penicillin and rifampicin. Antibiotic resistance rates to clindamycin, linezolid, erythromycin, tetracycline, cefoxitin, and ciprofloxacin were 82.5%, 78.9%, 73.7%, 63.2%, 33.3%, and 15.8% respectively. Antibiotic resistance genes were detected using primer-specific PCR and it was found that the prevalence rates of tetM, aac(6′)–aph(2″), mecA, tetK, ermc, and blaZ genes were 66.7%, 40.4%, 40.4%, 38.6%, 24.6%, and 3.51% respectively. Statistical analysis revealed significant (p < 0.05) relationships between the tetM, aac(6′)–aph(2″), and ermC genes and all parameters tested. A significant correlation between the aac(6′)–aph(2″) gene and the tetM (0.506) and ermC (−0.386) genes was identified. It was found that 23 (40.3%) S. aureus isolates were mecA positive, of which 10 (52.6%) out of 19 cefoxitin-resistant isolates were mecA positive and 13 (35.1%) out of 37 cefoxitin-sensitive isolates were mecA positive. The results of the present study demonstrated the detection of methicillin and multidrug resistant S. aureus isolated from samples obtained from wild pigeons and houseflies in the surroundings of a local public hospital in the Greater Durban area in South Africa. The findings of the study may account for the emergence of multidrug-resistant staphylococcal infections. The findings highlight the significant role of wild pigeons and houseflies in the spread of drug-resistant pathogenic S. aureus including MRSA. The conclusions of the present study highlight the improtant role of wildlife and the environment as interconnected contributors of One Health.

Isolation and Characterization of Lytic Bacteriophage against Methicillin Resistant Staphylococcus aureus from Pyoderma in Dog

Background: Pyoderma in dog poses complexity owing to involvement of multi drug resistant bacterial infection particularly Staphylococcus species. This may result into failure of antibiotic therapy in pyoderma. Therapeutic use of lytic bacteriophage (phage) may be an alternative to deal antimicrobial resistance in veterinary medicine. There is no report available on isolation and characterization of bacteriophage from clinical cases of pyoderma in animals, hence the study was performed with aim to isolate and characterize lytic phage against methicillin resistant S. aureus (MRSA). Methods: The phage was isolated from pus sample of dog suffering from severe pyoderma. The phage was isolated using MRSA as a bacterial host and was named as Staphylococcus phage BVC1 (SPBVC1). The morphology of the phage was determined using TME and was characterised by determining the host range and lytic potential of the phage at range of temperature and pH. Result: The morphology of phage revealed an icosahedral head of diameter 81.31 nm. with sheath and a central tube and a tail of 92.08 nm. It showed strong lytic activity against Methicillin resistant S. aureus and was stable under a range of temperature varying from 4oC to 45oC and pH from 4 to 11. The phage has shown lytic activity against the MRSA however no lytic activity against the MSSA was shown by the phage. The high specificity of the phage for MRSA indicated its potential use as an alternative therapeutic approach against multidrug resistant staphylococcal infections.

Anti-proliferative and antibacterial activity of oleo-gum-resin of Boswellia serrata extract and its isolate 3-hydroxy-11-keto-β-boswellic acid

Since time immemorial Boswellia serrata has been a useful traditional plant and its oleo-gum-resin is regularly used as an important constituent in herbal medicines for inflammation, cancer sufferers and cosmetics. This study focuses on the antibacterial and anticancer activities of B. serrata oleo-gum-resin extract, fractions and isolates. Time kill kinetic assay, combination study and ethidium bromide efflux pump inhibitory assay of the leading molecule 3-hydroxy-11-keto-β-boswellic acid (KBA) have been evaluated. The extract, fractions and the isolated molecule have shown anti-proliferative activity in different cancer cell lines, and the inhibition ranged from 13% to 93%. KBA caused anti-proliferative dose-dependent impacts on all human malignant cells (IC50 4.10–37.9 µg/ml). In antibacterial study of the extract, fractions and isolated molecules have shown minimum inhibitory concentration MIC range from 31.25 to 1000 μg/ml against 8 different Gram-positive and Gram-negative bacterial strains. KBA exhibited synergistic effect with a oxacillin against different drug resistant clinical isolates with fractional inhibitory concentration index (FICI) in the range of 0.325–0.450 µg/ml. KBA exhibited bacteriostatic killing mechanism and could also modulate ethidium bromide efflux pump to find its potential in combination therapy against multi-drug resistant Staphylococcal infections. KBA in the extract was quantified using HPLC-PDA and its structure was validated for the first time by X-ray crystallography. This study reinforced the promising treatment of KBA for cancer and S. aureus infections. KBA can be further explored to develop potential lead combinations of anti-cancer and anti-Gram-positive bacterial herbal formulations.

A recombinant fungal defensin-like peptide-P2 combats multidrug-resistant Staphylococcus aureus and biofilms.

There is an urgent need to discover new active drugs to combat methicillin-resistant Staphylococcus aureus, which is a serious threat to humans and animals and incompletely eliminated by antibiotics due to its intracellular accumulation in host cells, production of biofilms, and persisters. Fungal defensin-like peptides (DLPs) are emerging as a potential source of new antibacterial drugs due to their potent antibacterial activity. In this study, nine novel fungal DLPs were firstly identified by querying against UniProt databases and expressed in Pichia pastoris, and their antibacterial and anti-biofilm ability were tested against multidrug-resistant (MDR)S. aureus. Results showed that among them, P2, the highest activity and expression level, showed low toxicity, no resistance, and high stability. Minimal inhibitory concentrations (MICs) of P2 against Gram-positive bacteria were < 2μg/mL. P2 exhibited the potent activity against intracellular MDR S. aureus (bacterial reduction in 80-97%) in RAW264.7 macrophages. P2 bound to/disrupted bacterial DNA, wrinkled outer membranes and permeabilized cytoplasmic membranes, but maintained the integrity of bacterial cells. P2 inhibited/eradicated the biofilm and killed 99% persister bacteria, which were resistant to 100× MIC vancomycin. P2 upregulated the anti-inflammatory cytokine (IL-10) and downregulated pro-inflammatory cytokines (TNF-α/IL-1β) and chemokine (MCP-1) levels in RAW 264.7 macrophages and in mice, respectively. Five milligram per kilogram P2 enhanced the survival of S. aureus-infected mice (100%), superior to vancomycin (30mg/kg), inhibited the bacterial translocation, and alleviated multiple-organ injuries (liver, spleen, kidney, and lung). These data suggest that P2 may be a candidate for novel antimicrobial agents against MDR staphylococcal infections.

Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant Staphylococcal Infections by Antimicrobial-Immunomodulatory Duality.

Multidrug-resistant Staphylococcus aureus, including MRSA (methicillin-resistant) and VRSA (vancomycin-resistant), causes serious healthcare-associated infections, even sepsis and death. Here, we identified six novel cathelicidins (CATHPb1-6) from Python bivittatu, and CATHPb1 displayed the best in vitro pharmacological and toxicological profile. We further show that CATHPb1 exhibited evident protection in mice MRSA/VRSA infection models, given either 24 h before or 4 h after infection. The protection was all effective through different administration routes, but was blocked by in vivo depletion of monocyte/macrophages or neutrophils. CATHPb1 can rapidly and massively modulate macrophages/monocytes and neutrophils trafficking to the infection site, and potentiate their bactericidal functions. Meanwhile, CATHPb1 remarkably augmented neutrophil-mediated bacteria killing by facilitating neutrophil extracellular traps (NETs) formation and preventing its degradation. Acting through MAPKs and NF-κB pathways, CATHPb1 selectively enhanced the levels of chemokines while reducing the production of pro-inflammatory cytokines without undesirable toxicities. The much improved serum half-life and stabilities confer CATHPb1 an excellent prospect to become a novel therapeutic agent against multidrug-resistant staphylococcal infections.

Truncated Autoinducing Peptides as Antagonists of Staphylococcus lugdunensis Quorum Sensing.

Competitive quorum sensing (QS) antagonism offers a novel strategy for attenuating current multidrug resistant staphylococcal infections. To this end, a series of 10 truncated analogues based on the parent autoinducing peptides (AIPs) of Staphylococcus lugdunensis (groups I and II) and Staphylococcus epidermidis (groups I-III) were sequentially assessed against a newly developed Staphylococcus lugdunensis group I QS reporter strain. The truncated analogues based upon Staphylococcus lugdunensis AIP-1 (1) and AIP-2 (2) displayed respective IC50 values of 0.2 ± 0.01 μM and 0.3 ± 0.01 μM, while the truncated analogue of the Staphylococcus epidermidis AIP-1 (3) elicited an IC50 value of 2.7 ± 0.1 μM. These findings demonstrate the potential of cognate and "crosstalk" competitive quorum sensing inhibition using truncated AIPs as a means of attenuating staphylococcal infections in species beyond Staphylococcus aureus.

Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections.

Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections.

The prevalence of Inducible and Constitutive Clindamycin Resistance Among the Nasal Isolates of Staphylococci

One of the important sources of Staphylococci which causes nosocomial infections, is the nasal carriage of Staphylococci among Health Care Workers (HCWs). The commonest antibiotic which is preferred for the treatment of the methicillin and multi drug resistant Staphylococcal infections is clindamycin. The inducible clindamycin resistance in Staphylococci is not detected by the routine antibiotic susceptibility testing and it results in treatment failures. The present study was undertaken to know the prevalence of constitutive and inducible clindamycin resistance and its correlation with the methicillin resistance among the nasal isolates of Staphylococci which were obtained from different HCWs. Nasal swabs were collected from 206 HCWs and they were processed. The Staphylococci which were isolated were tested for methicillin resistance by using cefoxitin (30 μg) discs. The inducible clindamycin resistance was tested by using erythromycin (15 μg) and clindamycin (2μg) discs and the D test according to the CLSI guidelines. Inducible clindamycin resistance was seen in 21(16.40%) of the S.aureus and 14 (7.56%) of the coagulase negative Staphylococcal isolates. Constitutive clindamycin resistance was seen in 23(17.96%) of the S.aureus and 43(23.24%) of the coagulase negative Staphylococcal isolates. The inducible and constitutive clindamycin resistance was more common among the methicillin resistant Staphylococcal isolates. The prevalence of inducible and constitutive clindamycin resistance in the nasal Staphylococcal isolates which were obtained from the HCWs was high, especially among the methicillin resistant Staphylococci. The D test which is recommended by the CLSI should be routinely done to detect inducible clindamycin resistance, to prevent treatment failures.

In vitro Activity of Dalbavancin against Staphylococci Isolated in Istanbul, Turkey

Background: Staphylococci are a major cause of both community- and hospital-acquired infections. Dalbavancin is a new bactericidal second-generation lipoglycopeptide antibiotic to use in the treatment of multidrug-resistant staphylococcal infections. It is important to determine its activity against staphylococci isolated in Turkey. Dalbavancin has not yet been used in antimicrobial therapy in our country. Method: Dalbavancin was tested against a total of 453 staphylococcal strains by using the reference broth microdilution method. Organisms tested included: methicillin-resistant Staphylococcus aureus (MRSA; 237 strains), methicillin-susceptible S. aureus (MSSA; 144 strains) and methicillin- resistant coagulase-negative staphylococci (MR-CoNS; 72 strains). Results: MIC₅₀ and MIC₉₀ values of dalbavancin against MRSA, MSSA and MR-CoNS were found as ≤0.008 and 0.25 mg/l; 0.016 and 0.125 mg/l; and 0.016 and 0.5 mg/l, respectively. The overall distribution of dalbavancin MIC values ranged from ≤0.008 to 2 mg/l. If the interpretive breakpoint MIC value is ≤1 mg/l, the susceptibility rates of MRSA, MSSA and MR-CoNS strains were determined as 99.6, 100 and 98.6%, respectively. Conclusion: The MIC results for dalbavancin have demonstrated a good activity against both methicillin-sensitive and -resistant staphylococci isolated from hospitalized patients.

Surmounting antimicrobial resistance in the Millennium Superbug: Staphylococcus aureus

Abstract Staphylococcus aureus is the third most dreaded pathogen posing a severe threat due to its refractory behavior against the current armamentarium of antimicrobial drugs. This is attributed to the evolution of an array of resistance mechanisms responsible for morbidity and mortality globally. Local and international travel has resulted in the movement of drug resistant S. aureus clones from hospitals into communities and further into different geographical areas where they have been responsible for epidemic outbreaks. Thus, there is a dire necessity to refrain further cross movement of these multidrug resistant clones across the globe. The plausible alternative to prevent this situation is by thorough implementation of regulatory aspects of sanitation, formulary usage and development of new therapeutic interventions. Various strategies like exploring novel antibacterial targets, high throughput screening of microbes, combinatorial and synthetic chemistry, combinatorial biosynthesis and vaccine development are being extensively sought to overcome multidrug resistant chronic Staphylococcal infections. The majority of the antibacterial drugs are of microbial origin and are prone to being resisted. Anti-staphylococcal plant natural products that may provide a new alternative to overcome the refractory S.aureus under clinical settings have grossly been unnoticed. The present communication highlights the new chemical entities and therapeutic modalities that are entering the pharmaceutical market or are in the late stages of clinical evaluation to overcome multidrug resistant Staphylococcal infections. The review also explores the possibility of immunity and enzyme-based interventions as new therapeutic modalities and highlights the regulatory concerns on the prescription, usage and formulary development in the developed and developing world to keep the new chemical entities and therapeutic modalities viable to overcome antimicrobial resistance in S. aureus.

IN VITRO ACTIVITY OF ANTIMICROBIAL AGENTS AGAINST OXACILLIN RESISTANT STAPHYLOCOCCI WITH SPECIAL REFERENCE TO STAPHYLOCOCCUS HAEMOLYTICUS

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.

&lt;i&gt;In vitro&lt;/i&gt; activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to &lt;i&gt; Staphylococcus haemolyticus&lt;/i&gt;

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.

FUSIDIC ACID INDUCED ACUTE IMMUNOLOGIC THROMBOCYTOPENIA

Fusidic acid is used in hospitals as second-line therapy for multidrug-resistant staphylococcal infections. We report the first fully documented case of fusidic acid induced thrombocytopenia, in a 48-year-old patient. The thrombocytopenia was abrupt and severe but resolved spontaneously 7 d after drug withdrawal. The thrombocytopenia transiently relapsed 6 d later, when fusidic acid was reintroduced. Haemorrhagic signs were observed, but no severe bleeding occurred. Platelet transfusions failed to increase the platelet count. We detected an IgG platelet antibody in the patient's serum, that specifically recognized platelet glycoprotein IIb/IIIa only in the presence of fusidic acid. Fusidic acid induced thrombocytopenia should be considered as a possible cause for the thrombocytopenia frequently seen in the intensive care setting.