Plasmid Curing Analysis of Antibiotic Resistance in β-lactamase Producing Staphylococci from Wounds and Burns Patients
Hospitals worldwide are facing unprecedented crisis due to increasingly rapid emergence and dissemination of antimicrobial resistant staphylococci in wounds and burns and its environs via plasmid mediation. This study was conducted to evaluate the plasmid-mediated or chromosomal-mediated resistance in staphylococci. One hundred clinical swabs from wounds and burns patients were demonstrated for presence of staphylococci using mannitol salt agar. Various biochemical, DNase and β-lactamase test was carried out and the plasmid curing assay was demonstrated using 0.1 mg mL-1 acridine orange on antibiotic resistant isolates. The results revealed S. aureus (47) and coagulase negative staphylococci (CoNS) (6). β-lactamase producing species of S. aureus were 14 and CoNS was 1. Most isolates showed high resistance pattern to gentamicin, ciprofloxacin, norfloxacin, rifampicin, chloramphenicol, ampiclox and others. The antibiotic resistance isolates were highly indicative of plasmid-borne and few are chromosomal-borne after the plasmid curing analysis. The plasmid-mediated resistance observed among various antibiotics poses difficulty in treatment for clinicians. This high plasmid-mediated resistance among the isolates and from other studies calls for an urgent surveillance and epidemiological studies to infection control.
Received: March 13, 2013;
Accepted: April 13, 2013;
Published: November 21, 2013
Staphylococcus aureus is an important pathogen that causes skin, wound
and burn infections, septicaemia and endocarditis, such that infections involving
antibiotic resistant strains may impact on human health (Ombui
et al., 2000; Adegoke and Okoh, 2011; Yahmeen
et al., 2010; Bashir et al., 2007).
Coagulase-negative Staphylococcus (CoNS), a component of the normal skin
flora has become an important opportunistic pathogen in foreign body sites causing
both nosocomial and community acquired infections. Most developed countries
have reported an increase in colonization and infection in hospitalized patients
by CoNS while there are scanty data in developing countries (Akinjogunla
and Enabulele, 2010).
Since the introduction of antimicrobials, bacteria have developed mechanisms
for resisting the effects of antibiotics. The emergence of multidrug resistance
in Gram positive bacteria (Pneumococci, Enterococci and Staphylococci)
is a particularly important development (Bashir et al.,
2007). The levels of antibiotic resistant infections in the developing world
have increased steadily in the last few decades as a result of combination of
microbial characteristics and the selective pressure of antimicrobial use (Blondeau
and Tillotson, 2002; Ombui et al., 2000).
Microorganisms mechanisms of overcoming the activities of antimicrobial agents
include the production of structure-altering or inactivating enzymes (β-lactamase
or aminoglycoside-modifying enzymes), alteration of penicillin-binding proteins
or other cell-wall target sites, altered DNA gyrase targets, permeability mutations,
active efflux and ribosomal modification (Akinjogunla and
Enabulele, 2010; Gad et al., 2010).
Plasmids, which are extra-chromosomal materials, allow the movement of genetic
material, including antimicrobial resistance genes between bacterial species
and genera. It may contain resistance genes for single or multiple antimicrobial
agents, which have been reported among staphylococci to several therapeutically
useful antibiotics including penicillin family, streptomycin, rifampicin, fusidic
acid and novobiocin and fluoroquinolones (Akinjogunla and
Enabulele, 2010; Ombui et al., 2000).
The clinical significance of CoNS strains has emerged by their resistance characteristics
to methicillin and almost all classes of antimicrobial agents, therefore limited
treatment options and prolonged course of infection due to these CoNS species
could therefore have severe consequences (Akinkunmi and
Lamikanra, 2010; Ako-Nai et al., 2005). However,
plasmid profiles have been found useful in epidemiological surveillance of disease
outbreaks and in tracing antibiotic resistance patterns. Thus this study was
designed to determine plasmid-mediated resistance isolates on staphylococci
and the curing rate on each antibiotic tested.
MATERIALS AND METHODS
Collection samples: One hundred clinical wounds and burns samples were
obtained from different sites in patients undergoing dressing of wounds and
burns at General Hospital, Ekpan, Delta State, Nigeria. Exudates were obtained
from the infected sites of each patient with sterile cotton wool swabs and applied
to freshly prepared Mannitol Salt Agar (MSA) and Nutrient agar (Oxoids). The
cultures were then transferred to the laboratory where they were incubated at
37°C for 24 h.
Isolation of Staphylococci and species identification: Colonies growing
on nutrient agar slants were streaked on top of freshly prepared plates of MSA
and incubated at 35°C for 24 h. Primary characterization of isolates was
based on the gram stain, morphological and cultural characteristics, fermentation
on MSA, catalase and coagulase (tube) test and DNase agar test. The ability
of each S. aureus and CoNS isolates to elaborate penicillinase was evaluated
using the method described by Ako-Nai et al. (2005).
The pure colonies were further sub-cultured and stored on nutrient agar slants
at -20°C for further analysis.
β-lactamase assay: This test was carried out as described by Ako-Nai
et al. (2005). Strips of starch paper measuring 4x7 cm were cut and
sterilized with 70% ethanol. These strips were then soaked for 10 min in a solution
of benzyl penicillin dissolved in phosphate buffer containing 105
units. They were spread over an area of 2 to 3 mm. Each test paper was then
used to test two organisms at time with the inocula placed at least 2 cm apart.
The Petri dishes were then incubated for 30 min at 37°C after which the
plate was flooded with Grams iodine solution. This was immediately drained off.
This caused the starch paper to turn uniformly black within 30 sec of application.
Colonies with decolourized zones thereafter were indicative of β-lactamase
production. Results were read within 5 min as black background tends to decolourize,
making interpretations more difficult.
Determination of antibiotic resistance profile: Wounds and burns isolates
were subjected to antibiotic screening by disk diffusion method as described
by CLSI (2008). Inocula were prepared by diluting overnight
cultures in sterile NaCl (0.9%) suspension and then marched with the McFarland
turbidity index. Bacterial suspensions were then plated on to Mueller Hinton
Agar and the commercially available antibiotic discs were placed on lawn of
culture and the plates incubated overnight at 37°C. Sensitivity, intermediate
and resistance were determined by the zone of complete growth inhibition around
each disk according to reference standards. Reference type S. aureus
strain (ATCC 25923) was used as positive control.
Plasmid curing analysis: Plasmid curing was carried out in order to
determine the location (plasmid-borne or chromosomal) of the drug resistance
marker(s). The curing (elimination) of the resistant plasmids of the S. aureus
and CoNS isolated was done using sub-inhibitory concentration of 0.10 mg mL-1
of acridine orange as described by Rasool et al. (2003),
Yah et al. (2007) and Akortha
and Filgona (2009) with slight modification. Isolates were grown for 24
h at 37°C in Mueller-Hinton broth containing 0.1 mg mL-1 acridine
orange. The broth was agitated to homogenize the content and loopful of the
broth medium were cultured on MHA plates and antibiotic sensitivity testing
was carried out as previously described. Absence of zone of inhibition on Mueller
Hinton agar was indicative of plasmid-mediated resistance (plasmid cured) while
presence of zone of inhibition on Mueller Hinton agar was indicative of chromosome-mediated
(plasmid not cured).
This study revealed a high isolation rate of Staphylococcus spp. 53(53%)
with 47 isolates as S. aureus and 6 isolates as CoNS. Of the 47 S.
aureus isolates, 14(30%) were β-lactamase producing species and 33(70%)
were non-β-lactamase producing species. Whereas, only 1 (17%) β-lactamase
producing species was indicated of the 6 CoNS isolates. The characterization
test with DNase agar showed 38(72%) isolates of staphylococci as positive and
15(28%) isolates as negative (Table 1, 2).
Most of the antibiotics employed showed high resistance pattern against the
tested staphylococci. 46 S. aureus was tested against gentamicin, 28
(60%) isolates were resistant, 15 (32%) sensitive and 3 (9%) intermediate while
all CoNS (100%) assayed showed resistance. The staphylococcal isolates tested
against the fluoroquinolones used in this study showed high resistance pattern
in Norfloxacin (62%), Ciprofloxacin (57%) and Levofloxacin (51%) in S. aureus
while in CoNS, the resistance pattern in norfloxacin, ciprofloxacin and levofloxacin
are 67, 100 and 83%, respectively (Table 3). The plasmid curing
analysis on resistant isolates as revealed in Table 4 showed
that most resistant isolates were cured (plasmid-borne) and few isolates were
resistant against tested antibiotics after curing (chromosomal-borne) as compared
with the initial resistant pattern before curing (pre-curing).
||Biochemical characterization and identification of wounds
and burns isolates from hospital patients in accident and emergency unit
||Characterization and occurrence of β-lactamase (βL)
and non-β-lactamase producing S. aureus and CoNS isolated from
wounds and burns patients
Several studies have revealed an increase in antibiotic resistance pattern
associated with S. aureus and more importantly on CoNS, which has equally
being posing a great health challenge to humans and animals and it was also
observed in this study (Bashir et al., 2007;
Ombui et al., 2000; Yahmeen
et al., 2010; Akinkunmi and Lamikanra, 2010;
Akinjogunla and Enabulele, 2010). This trend of increase
in antibiotic resistance was also revealed in a study on hospital currency notes
by Adegoke and Okoh, 2011. In determining the mechanism
of resistance to antibiotics by staphylococci, β-lactamase production and
plasmid curing assay was conducted. We observed a fairly high level of β-lactamase
production in staphylococcal isolates especially among the S. aureus.
High levels of β-lactamase production in Staphylococci had also been reported
(Gad et al., 2010; Adegoke
and Okoh, 2011; Akinjogunla and Enabulele, 2010;
Ako-Nai et al., 2005). The plasmid curing assay
revealed that most of the antibiotic resistant staphylococci isolated in this
study were plasmid-mediated since 56-91% of the isolates showed zones of inhibition
(cured) when tested against the selected antibiotics while 2.6-31% showed zones
of inhibition (plasmid not cured) indicating chromosomal-borne resistant gene.
The screening of the isolates with acridine orange resultantly suggest that
the resistance markers were stably lost, which is in line with that of previous
studies (Akinjogunla and Enabulele, 2010; Adegoke
and Okoh, 2011; Gad et al., 2010; Yah
et al., 2007; Akortha and Filgona, 2009).
||Antibiotic susceptibility profile on isolated Staphylococcus
spp. from wounds and burns hospital patients
||Plasmid curing analysis of resistant staphylococci isolated
from wounds and burns patients with Acridine orange (0.1 mg mL-1)
This study had examined the incidence of staphylococci, its susceptibility
patterns to different antibiotics and the drug resistance factor mediated by
β-lactamases and plasmid-borne genes. Thus indicating that most resistance
to antibiotics is plasmid-mediated, this can easily be transferred from one
strain to another or from one organism to another within the same environment.
However, this study intends to carry out the molecular study on the plasmid-borne
and chromosomal-borne gene associated with antibiotic resistance. Therefore,
hospital and community management of infection control and surveillance of antibiotic
resistance is highly and urgently necessary.
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