Key-words:
Antimicrobial susceptibility testing - Brack hospital - coagulase-negative staphylococci
- Libya - multidrug-resistant staphylococci
Introduction
Staphylococcus species are major healthcare-associated pathogens responsible for critical and opportunistic
infections among humans associated with increased morbidity and mortality rates.[[1]] Methicillin-resistant staphylococci (MRS) are bacterial pathogens expressed by
variable Staphylococcus species showing significant multidrug resistance to important antimicrobial classes
including drugs of last resort such as glycopeptides.[[1]] MRS represent serious medical and public health concerns that rapidly spread with
variable epidemiological distribution worldwide.[[2]]
In Libya, methicillin-resistant Staphylococcus aureus (MRSA) is the most reported nosocomial pathogen exclusively isolated from human healthcare
settings; however, most of the available data are inadequate, and mainly originated
from urban areas (i.e., cities from the north coastal region of Libya) with a paucity
of information from suburban and underdeveloped areas.[[3]],[[4]] From the south region of Libya, only two studies have documented healthcare-associated
organisms (i.e., MRSA and Pseudomonas aeruginosa) isolated at Sebha medical center.[[5]],[[6]] Recently, MRS have been reported from humans and companion animals, mainly belonging
to the species of coagulase-negative staphylococci (CoNS) presenting public health
and zoonotic concerns.[[7]],[[8]] The current study investigated a collection of 110 identified staphylococci strains
isolated from humans from various clinical and nonclinical sources at Brack hospital
in the southern region of Libya between August and December 2018. The collected isolates
were further analyzed to identify and confirm the genus and species of the isolates
and further characterize the antimicrobial susceptibility profiling. Brack hospital
is a major healthcare setting that provides various medical and health services to
the community in the southern region of Libya.
Materials and Methods
Source of the collection
The collection was isolated from patients (n = 63) and healthcare works (n = 47) and obtained from nasal swabs (n = 23), hand swaps (n = 73), and clinical samples from urine catheters (n = 14). The collection originated from 79% (n = 87) of females and 21% (n = 23) of males, and the age of the involved individuals ranged from 1.5 to 63 years
(mean = 28.5 years). Isolates were identified based on standard laboratory procedures
using direct culturing onto blood and MacConkey agar plates and further identified
based on the typical criteria of staphylococci species including Gram-positive cocci,
clustering, and catalase reactivity. Isolates were stored at −20°C until further analysis.
Laboratory identification and biochemical characterization
Each isolate was enriched in brain heart enrichment broth and incubated aerobically
for 24–48h at 37°C. A loopful from each broth was streaked onto both mannitol salt
and blood agar and incubated for 24h at 35°C. Plates were then checked for typical
growing colonies featuring staphylococci as yellow, circular, and shiny colonies.
A typical colony was selected from each plate and further examined with a Gram stain
and catalase test and identified as presumptive staphylococci. Isolates were further
tested with a BD Phoenix automated identification and susceptibility testing system
(PAMS, MSBD Biosciences, Sparks, MD, USA) for definite characterization at the genus
and species levels and to determine the susceptibility against antimicrobial agents.
The antimicrobial susceptibility profile was identified based on the interpretation
of the Phoenix system and by the criteria of CLSI guidelines.[[9]] The detection of MRS was based on the interpreted criteria of minimum inhibitory
concentration (MIC) for oxacillin and cefoxitin as follows: Susceptible, MIC ≤2 μg/ml,
and resistant, MIC ≥4 μg/ml. The MIC breakpoints for CoNS (excluding S. lugdunensis) and S. pseudintermedius were ≤0.25 mcg/ml for susceptibility and ≥0.5 mcg/ml for resistance.
Results
In total, only 57.5% (n = 42 of 73) were confirmed as species and subspecies represented by ten different
staphylococci species: nine species of a space after CoNS. (n = 32; 76.2%) and one subspecies of (CoPS) (n = 10; 23.8%) [[Table 1]]. The identified species were S. aureus (n = 10), S. gallinarum (n = 6), S. xylosus (n = 6), S. saprophyticus (n = 4), 2; S. epidermidis (n = 4), S. warneri (n = 4), S. equorum (n = 2), S. simulans (n = 2), S. kloosii (n = 2), and S. hominis (n = 2), [[Table 1]]. Furthermore, 16.6% (n = 7 out of 42; 6 CoNS and 1 CoPS) of the strains expressing MRS phenotypes were found
to have similar antibiogram profiles [[Table 2]]. The remaining isolates were susceptible to all antimicrobial classes.
Table 1: Characterization of Staphylococcus species (n=42)
Table 2: Antimicrobial susceptibility profiling of methicillin-resistant staphylococci expressing
strains (n=7)
The seven MRS isolates were respectively distributed between S. gallinarum (n = 3), S. xylosus (n = 2), S. aureus (n = 1), and S. equorum (n = 1). Of these, five MRS isolates expressed typical resistance to penicillin, oxacillin,
ampicillin, amoxicillin–clavulanate, cefoxitin, and cefotaxime but were susceptible
to gentamicin, tetracycline (except one), trimethoprim–sulfamethoxazole, nitrofurantoin,
moxifloxacin, rifampin, ciprofloxacin, linezolid, daptomycin, teicoplanin, vancomycin,
fusidic acid, erythromycin, clindamycin, and mupirocin. Two MRS S. xylosus isolates expressed MLSBi phenotype expressing further resistance to erythromycin,
clindamycin, trimethoprim, and tetracycline [[Table 2]].
Discussion
In general, most of the available information on Staphylococcus from Libya has focused on S. aureus of clinical sources with little information on other species. A previous study investigated
a collection of 218 isolates of staphylococci originated from clinical samples collected
at Tripoli hospital reported MRSA in 28.4% (n = 62/218) of the collection followed by MRCoNS (21.5%; n = 47/218).[[10]] Another study involving different hospitals in Benghazi reported an MRSA prevalence
at 8% of samples collected from various surfaces and environmental sources and identified
32 out of 100 S. aureus strains expressing the MRS phenotype but susceptible to vancomycin and mupirocin.[[11]]
In the current study, 42 isolates were confirmed as Staphylococcus species of which seven isolates expressed MRS phenotypes predominantly of the CoNS
group originating from nasal and hand samples. A previous study from the southern
region of Libya investigating 43 strains of S. aureus recovered from different departments at Sebha medical center reported MRSA in 16%
of the isolates but susceptible to vancomycin.[[5]] The revelation of the present study showed the variable epidemiological status
of Staphylococcus species in the studied area but may also indicate an epidemiological shift in the
distribution of Staphylococcus species within the Libya healthcare system. In fact, a recent molecular investigation
on a collection of clinical S. aureus collected at the largest Libyan hospital in Tripoli revealed the presence of atypical
genotypic strains among MRSA strains showing a dynamic molecular shift in MRSA consistent
with global molecular changes.[[3]] Nevertheless, the available information on the epidemiological distribution of
Staphylococcus species within the Libyan health system and the community is incomplete and inadequate
mainly due to underdeveloped infrastructures and economic resources.
S. aureus and S. epidermidis are typical colonizers of the skin and nares, linked to biofilm formation and responsible
for serious persistent infections.[[12]],[[13]] CoNS, on the other hand, are emerging opportunistic organisms able to persist on
a variety of environmental surfaces but less involved in community-associated diseases.[[14]] The majority of the CoNS species identified in the current study are typical human-associated
organisms (i.e., the S. epidermidis-like group – S. epidermidis, S. haemolyticus S. capitis, S. hominis) and more commonly isolated from opportunistic and bloodstream infections associated
with antibiotic use and the use of medical devices.[[14]],[[15]]
Staphylococci of animal and farms origins are frequently associated with human and
bloodstream infections showing multidrug resistant to important antimicrobial classes
(e.g., glycopeptides and fluoroquinolones).[[16]],[[17]] Of these, S. gallinarum, known to be widespread in the environment and isolated from skin and respiratory
tract of farm animals, is increasingly reported from opportunistic infections associated
with the prevention use of antibiotics.[[16]] S. xylosus is a commensal organism of the skin and the mucous membranes of humans and animals
responsible for opportunistic and zoonotic infections (e.g., mastitis or dermatitis).[[18]] This species is frequently isolated from animal products (e.g., cheese, milk and
meat) and used in the development of flavor and food processing due to its antioxidant
and degradation properties.[[19]] The identification of such animal- and environmental-associated species raises
concerns over zoonotic contamination and contacts.[[20]]
MRSA and MRCoNS are commonly isolated from humans and animals responsible for severe
infections in healthcare facilities and the community.[[21]],[[22]] MRCoNS are an emerging cause of hospital-acquired infections; however, the available
knowledge on their prevalence is very limited from the underdeveloped regions.[[14]],[[23]] In the current study, six MRCoNS were identified showing similar multidrug-resistance
properties (i.e., AMP, Pen, AMX, OXA, FOX, CTX) of which three were S. gallinarum and expressed further and variable intermediate susceptibility to erythromycin, clindamycin,
and teicoplanin. In addition, two MR S. xylosus strains were characterized expressing MLSB phenotype showing further resistance to
trimethoprim–sulfamethoxazole and tetracycline which reportedly linked to antibiotics
usage.[[24]]
Nasal/nasopharyngeal colonization with MRCoNS is documented as a major risk factor
for persistent and drug-resistant infections.[[23]],[[25]] CoNS are recognized as a major reservoir of virulent and antibiotic resistance
genes that can be acquired by other staphylococci mainly through the transconjugant
transfer of the staphylococcal cassette chromosome mec (SCCmec) transposon containing the mecA gene, as in the case of transfer between S. aureus and S. epidermidis.[[26]] Another mec gene homolog is mecC, which has about 70% comparability with the mecA gene, and can be carried by SCCmec elements and isolated from animals, human clinical specimens, and the environment.[[27]] Reportedly, the identification of the high rate of MRCoNS, particularly those carrying
the mec genes among human isolates, may reflect higher exposure to antimicrobial drugs and/or
the coexistence of resistance determinants. This may favor the horizontal transfer
of mobile genetic elements to other commensal organisms leading to the emergence of
more virulent drug-resistant strains such as vancomycin-resistant S. aureus.[[28]],[[29]] Unfortunately, due to the limitations of the current study, these important genes
(i.e., mecA, mecC, and pvl) were not investigated.
As the global epidemiological distribution of staphylococci and the associated multidrug
resistance phenotypes have dramatically changed, accurate laboratory identification
and characterization is important.[[3]],[[30]] The Phoenix system proved to be efficient for accurate identification and antimicrobial
susceptibility of staphylococci including CoNS as well as other major healthcare-associated
pathogens such as enterococci and Gram-negative rods.[[31]] Strains of S. aureus may also be misinterpreted using phenotypic testing methods and microbiological automated
systems with other CoPS or certain clinically important CoNS strains such as S. lugdunensis and S. schleiferi.[[32]] Therefore, reliable, efficient, and advanced molecular laboratory methods such
as MALDI-TOF MS or polymerase chain reaction technology are required.[[33]]
Conclusion
This study revealed novel information on important healthcare-associated pathogens
isolated from a healthcare setting in the southern region of Libya. The current investigation
revealed a high rate of CoNS among the collection expressing concerning MRS phenotype
underlying the importance of monitoring all Staphylococcus species. Appropriate and effective prevention strategies in healthcare facilities,
including antibiotics stewardship and epidemiological studies, are required to control
the dissemination of MRS.
