Infection Control of Spatial Disseminated Multi-Antibiotics Resistant And Phylo-Diverse Staphylococcus Aureus Pathotypes


 Focal dissemination of multi-antibiotic resistant (MAR) Staphylococci pathotypes regulated by agr functionalities was investigated and evaluated for infection control. Non-repetitive Staphylococcus aureus strains from soft and skin infections disseminated in several communities were recovered and biotyped, assayed for biofilm and profiled for antibiotic resistance. Strains were further genotyped for spa types, virulence and resistant genes; and mapped for geospatial distribution. Clonal diversity and functional accessory gene regulators (agr) were also evaluated. Staphylococcal infection was not significant with age group (p>0.05), but high rate of MSSA (53.0%) and MRSA (1.5%) was observed. Median resistance rates were significantly differ (p=0.001) but highest 75 th percentile and media resistance rates were observed in wound infection. Resistance rate of 78.8% at MIC 50 32µg/ml and MIC 90 128µg/ml to amoxicillin-clavulanate, and more than 40% resistance to ceftazidime, ciprofloxacin, gentamycin, ofloxacin, sulfamethoxazole and tetracycline with MIC 90 and MIC 50 at 32 µg/ml were observed. More than 0.83 multi-antibiotic resistance index (MARI) were observed among the strains that clustered into separate phylo-group expressing high beta- lactamase and strong biofilm production. Heterogeneous spa types t442 (wound and pus), t657 (wound), t091 (ear) and t657 (ear and wound) revealed high phylo- diversity. Only 4.6% pvl + MSSA-CC1 agr I, pvl+ MSSA-CC5 (13.6%) and pvl+ MRSA-CC7 agr II (4.6%), expressed enterotoxin; sea, sec, sed, sej, Leukocidins ( LukF-PV, lukD, lukE ), proteases (aur, slpA sspB, sspE ) and resistance genes (fosB, msr (A), bla mph(C),aphA3, sat, fosB, sdrM, Q7A4X2) . Phylogenetic related spa types of livestock origin, specifically bovine milk clustered with detected strains that were prevalent in urban communities with focal dissemination to other nearest suburbs. Clonal dissemination resistant pvl+MAR MSSA-CC1 and MRSA- CC5 encoding agr were predominant in several peri-urban communities. This require adequate geno-surveillance, population-target antimicrobial stewardship, extensive community health care intervention policy and well-structured infection control programs to prevent further focal dissemination.


INTRODUCTION
Staphylococcal infection remains a major health challenge in several countries, with a huge resultant adverse effect ranging from life-threatening diseases such as pneumonia, bacteremia to high mortality cases [1]. Several clonal complexes have been reported from different regions of the globe [2], while various spa types kept evolving with diverse genomic recombination, phylogenetic clones, and repeated nucleotide mutations, giving rise to fatal virulent strains [3]. In addition, there is a capability of numerous clonal strains of Staphylococcus aureus to adapt by its specificity for colonization through production of poly-N-acetylglucosamine to produce biofilm needed to evade immune response and antibiotic activity [4].
Severity of staphylococci infection correlates with virulence expression which is regulated through the functionality of accessory gene regulators (agr), which encodes a two-component signal transduction system that could down-regulate surface proteins metabolism and up-regulate secreted proteins during in vitro growth [5], favoring the transcription of several secreted virulence factors (particularly enterotoxins, hemolysins, and TSST-1) [6]. Functional agr groups were reported to enhance persistent staphylococci bacteraemia and soft tissue tropism with low antibiotic susceptibility to penicillin, cephalosporin and vancomycin [7,8]. Similar clonal spread of MRSA (methicillin-resistant Staphylococcus aureus) and MSSA (methicillin-susceptible Staphylococcus aureus) is becoming pandemic in many several communities in Africa, mostly Nigeria where animal husbandry, behavioural responses and declined demographic factors enhance continuous dissemination of staphylococcal infection with high degree of antibiotic resistance [9]. The misuse and unregulated prescription of penicillin derivatives in high and uncontrollable proportion for treating several extra-intestinal infections such as abscess, ear infections, subcutaneous tissue inflammation, nasal discharges particularly in children and postsurgical wound culminated in a high rate of resistance and continuous development of methicillin-resistance strains [10,11].
Heterogenous spa types identified among several MSSA and MRSA carriers and infected subjects [12] require clonal diversity and staphylococcal infection surveillance, tracking and strains genotyping [13,14]. Moreover, a repeated evolution of various spa types has kept driving dynamics spread of staphylococcal infection that were demonstrated in various infection outbreaks, localized epidemics and community-acquired infections. Mapping the spread and dissemination of mecA gene among spa types is highly needed for reliable genomic tracking, localization and control of staphylococcal infection in several local communities with high-level dissemination and distribution of resistant spa types probably acquired from livestock [12].
In this study, we investigated the antibiotic resistance distribution and prevalence of agr groups of phylodiverse S. aureus strains characterized by various spa repeats and assessed the potential association between different agr group functionalities, severity and staphylococci infection controls.

METHODS
Isolates collection: Non-repetitive clinical samples totaling 256 including purulent pus (n=58), aspirates (n=34), wounds (n=55) and otitis media (n=36), eye infection (n=14), throat (n=35) and endocervical (n=24), collected between June 2017 and August 2018 from outpatients attending three major health facilities which serves as referral clinics in southwest Nigeria. Ethical permissions for the study were obtained and data on their gender, age, disease conditions and subjects' location of residence were not fully retrieved. Each sample were cultured for Staphylococci strains and phenotypically characterized on Baird-Parker agar and Mannitol salt agars, Gram stained for cellular morphology, tested for catalase and coagulase production as previously discussed [15] Phenotypical beta-lactamase detection and antibiogram: Beta-lactamase production was assayed with modified starch-acidometric method [16] and Minimum inhibitory concentrations (MICs) for each antibiotic class against the strain was determined using micro-broth dilution assay [17] with 12 panel antibiotics consisting of tetracycline, ceftazidime, ciprofloxacin, gentamycin, ampicillin, amoxycillin-clavulanic acid, cefuroxime, ofloxacin, sulfamethoxazole, erythromycin, penicillin, vancomycin and Linezolid. Phenotypic resistance was interpreted according to CLSI guidelines [18]. Phenotypic screening for methicillin resistance was further determined by assessment of Staphylococci growth on Mannitol salt agar and Mannitol salt agar supplemented with 4μg/ml Oxacillin as previously described [19].
Biofilm detection and mecA and pvl genotyping: Phenotypic assessment of biofilm production was done in micro-broth bioassay [20]. Extracted DNA template was genotyped for mecA gene using mec5 (AAAATCGATGGTAAAGGTTGGC) and mec6 (AGTTCTGCAGTACCGGATTTGC) primers (following previous described protocol and pvl gene with primers pvl-F (AATGAAATGTTTTTAGGCTCAAGACA) and pvl-R (TGGATAACACTGGCATTTTGTGA) [21]. Amplicon products were electrophoresed on 1.5% agarose gel. Multiantibiotic resistance index (MARI), degree of biofilm production, beta-lactamase production and mecA relatedness among the strains were evaluated with dendrogram analysis constructed with DendroUPGMA algorithm.
Genotyping and clonal diversity of spa types: Extracted genomic DNA obtained from overnight culture, was typed for S. aureus protein A (spa gene). PCR assay was performed in constituted reaction mixture of 2x MyTaq following manufacturer's instruction and the image of the array was recorded and analysed using a designated reader and software (Arraymate, Iconoclust, Alere Technologies) [25].
Geospatial analysis: Geographical coordinates of individual subjects with staphylococci infection were identified and recorded with differential global positioning system (GIS) and interpolated for analysis in ArcGIS programme with respect to land division according to boundary marks in southwest Nigeria [26].  Table 2).   (Figure 4).

DISCUSSION
Continuous spread of staphylococcal infection in several communities is now becoming a threat to the populace and mostly the children. Methicillin susceptible S. aureus infections are now commonly observed among the children with high risk of sores, blood stream infection, and scalded skin infection which are recorded due to low immunity, poor hygiene and possible transmission from Staphylococci-carrier mothers [27]. Occupation and routine activities of many young adults and men could be considered a pre-disposing risk factor. Data relating subject occupation with staphylococcal infection was not available but recorded MRSA and MSSA detection in wound largely suggest stemming increase and spread of community-acquired staphylococcal infections [28].
Nosocomial staphylococcal infection could not be ruled out as hospital infection control could be compromised due to low hygiene and staff carriage of multi-antibiotic resistance Staphylococci strains [29]. A significant low susceptibility was observed among the strains collection to ceftazidime, ciprofloxacin, amoxycillin-clavulanic acid and cefuroxime. Particularly strains from wound, ear, pus and aspirates showed a reflection of prolonged use and misuse of antibiotics in the treatment of staphylococcal infections. Continuous evolution and selective pressure of antibiotic resistance cannot be ruled out as a driven factor for the prevalence of resistant pathotypes across various population groups as evident with more than 40% resistance to tetracycline at MIC 90 (64µg/ml) and MIC 50 at (8 µg/ml).
The ability to treat multi-antibiotic resistant staphylococci strain characterized with biofilm is a challenging situation [30] and detection of different phylo-related strains expressing high level antibiotic resistance with potential to produce both biofilm and beta-lactamase enzymes put the populace at great risk [31]. Antibiotic resistance relatedness of several MSSA showing observable in-vitro biofilm production reflects acute systemic infection severity and pathology that could progress to high morbidity [32,33], making MSSA-biofilm producing strains in soft tissue and skin infections difficult to treat [34]. High biofilm production in deep layer secretions in cases of septic wound, tissue abscess and purulent pus exudates could reduce drug penetration, inflammatory response and impairment of cellular immune activity [35]. In addition, strains with high MARI beta-lactamase and high biofilm production are considered important pathotypes that needed to be designated for surveillance and assessment among diverse population at different localities. It is highly imperative to have periodic surveillance for these clusters with related resistance profile toward prevention of local sporadic outbreak and control of antibiotic misuse. However, unregulated prescription and abuse of antibiotics in several local communities in southwest Nigeria largely contribute to increase circulating resistant phylo-groups. Relative increase of resistant MRSA isolates to penicillin derivatives has been found to be associated with encoded mecA gene and betalactamase production which is a major factor to be considered towards achievable control of MRSA spread [36].
In addition, identification of heterogeneous spa types in extra-intestinal infections clearly showed high phylodiverse spa strains clustering into various different clades. In spite of this strain-diversity, profound relatedness with other meta-spa types suggests high level dissemination of similar clonal groups [37]. This is a clear evidence of involvement of spa types in single or multiple staphylococcal infections having high substantial impact through localization and distribution in soft tissue for adaptation, colonization and pathogenesis thereby initiating severe infection [38,39].
Identified phylo-diverse MSSA from Nigerian communities indicates active clonal transfer from other locations [40]. Detection of heterogeneous spa sequences from various skin and soft-tissue infections (wound, abscess and pus), is an evidence of genetic recombination of spa repeats from livestock-associated staphylococci particularly from bovine milk [41]. This further establish animal to human transfer which is observed in most communities where animal husbandry is usually practice within and around the households. Consumption of unpasteurised bovine milk, poor milk wastes disposal and frequent human contact with udder during animal milking are observable, is usually predisposing risk factors to be considered as major sources and spread of diverse spa strains with high level antimicrobial resistance traits [41]. It is also important to note that reported multi-antibiotic resistant MRSA identities in this study could perpetuate severity with little or no therapeutic options. Scratches, pecking and bite on human skin by poultry, cattle and other livestock cannot be ruled out as major contributor to animal clonal strains found among this populace. It is imperative to investigate mechanism of animal transfer of spa types to human and high prevalence of these associated livestock spa types. It is also necessary to evaluate the emerging animal clonal spa types vis-a-vis animal husbandry and antibiotic residue in milk in order to safeguard the populace and drastically reduce dissemination and risk of contracting antibiotic resistant strains.