| Abstract|| |
Background: Staphylococcus aureus is a major pathogen to man and is invasive when the skin is breached as well as in trauma. The frequency and transmission of S. aureus was determined in high vaginal swabs (HVS), oropharynx, and breast milk of human immunodeficiency virus (HIV)-seropositive women to their neonates at healthcare centres, Ondo State, Nigeria between November 2014 and December 2015. Materials and Methods: A total of 114 HIV pregnant women were screened for S. aureus isolates in the HVS, oropharynx, and breast milk employing sterile cotton tipped applicator. The swabs were subsequently introduced into sterile thioglycollate medium and incubated at 37°C for 24 h. When growth was noticed, a loopful was introduced onto freshly prepared mannitol salt agar (MSA). Colonies of cocci that fermented mannitol on MSA were presumptively deemed as S. aureus, but confirmed as S. aureus isolates by the slide and tube agglutination tests in pooled human plasma. HIV seropositivity was determined by the HIV1/2 strip (Determine Test, Alére, London, England, UK) and confirmed as such by enzyme-linked immunosorbent assay (Abbot laboratories, Chicago, USA). Phenotypes were determined by susceptibility testing and phylogenetic relatedness by random amplified polymorphic DNA polymerase chain reaction. Results: Results showed that 50 (43.85%) were colonized by S. aureus; however, 4 (8%) showed mother-to-child transmission from the three sites. Although all isolates tested were resistant to nalidixic acid and penicillin, 93.75% of isolates were sensitive to imipemen. Conclusion: The study revealed a high frequency of colonization of S. aureus in HVS (64%), oropharynx (20%), and breast milk (16%). Horizontal transmission from mother to neonate was 8% while phylogroups showed similarities in genetic relatedness.
Keywords: maternal-neonate transmission, Phenotypes and phylogenetic relatedness, Staphylococcus aureus
|How to cite this article:|
Ebhodaghe BI, Ako-Nai KA, Kassim OO. Phenotypic and phylogenetic relatedness of selected Staphylococcus aureus strains cultured from HIV seropositive mothers and their neonate's pair. Ann Trop Med Public Health 2017;10:384-90
|How to cite this URL:|
Ebhodaghe BI, Ako-Nai KA, Kassim OO. Phenotypic and phylogenetic relatedness of selected Staphylococcus aureus strains cultured from HIV seropositive mothers and their neonate's pair. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Nov 12];10:384-90. Available from: http://www.atmph.org/text.asp?2017/10/2/384/208729
| Introduction|| |
Staphylococcus aureus is a major pathogen to man. However, the organism displays a central epidemiological challenge in that it is also routinely found colonizing healthy individuals; thus, some strains have been known to enhance the potential to produce diseases. Approximately 20–40% of normal individuals harbor these microorganisms in their anterior nares. The key to unravelling the rate of acquisition and dissemination of S. aureus lies in understanding the carrier state, which is highly desirable in clinical medicine to control nosocomial infection. S. aureus nasal carriers are more susceptible to skin sepsis and postoperative infections caused by these organisms. S. aureus is a familiar resident of the nares, skin, and oropharynx in humans, and the prevalence rate varies from one region to another. Studies have shown that in the last 30 years in Ile-Ife and southwestern Nigeria the nasal carrier rates have fluctuated from 8% to 38%.,, S. aureus can colonize both apparently healthy individuals as well as immunocompromised individuals including human immunodeficiency virus (HIV) seropositive patients at late pregnancy to their newborns. Newborns of carrier mothers are at risk of acquiring S. aureus colonization. Most newborns of carrier mothers have been reported to be colonized within the first month of life, and horizontal transmission from mother is probably the major source for S. aureus carriage in newborns. Transmission of S. aureus is low, and the invasiveness of this organism is derived from toxin and biological active extracellular enzymes including alpha toxins, pyrogenic toxins, and cell wall protein A. Mother-to-child transmission of S. aureus can occur horizontally postpartum  when newborns are most vulnerable to infections mainly due to relatively immature immune system. Studies have shown that genetically identical strains have been isolated in 32/40 (80%) mother-newborn pairs and that the source of the newborn strains were maternal nasal strains 29/32 (90%).
Our study determined the frequency of colonization and mode of transmission of S. aureus isolates isolated from 114 HIV infected pregnant women aged 19–43 years (mean age of 31.81 years) from three sites, namely, high vaginal swabs (HVS), oropharynx, and breast milk samples of lactating mothers and anterior nares of their newborns/neonates. The phenotypes and phylogenetic relationship of these strains were also compared. Most studies on mother-to-child colonization with S. aureus have been reported in apparently healthy individuals, only a few have been reported in immunocompromised individuals, hence this study was conducted.
| Materials and Methods|| |
HIV screening among cohort
A 5 mL volume of whole blood was collected in a sterile vacutte EDTA tubes K3 using sterile 38 × 0.8 mm needles from each participant, and a small aliquot was applied onto the HIV-1/2 strip (Determine Test, Alére, London, England, UK) for the preliminary determination of HIV serostatus. Confirmatory test for HIV infection was performed using the Abbott enzyme-linked immunosorbent assay (ELISA) procedure (Abbott Laboratories, Chicago, IL, USA).
Criteria for study inclusion
Participants were pregnant women in the third trimester of pregnancy, fully registered at the antenatal clinics of the selected healthcare centres, with no restriction by age. Participants were also encouraged to attend all physician appointments throughout the study. Patients who did not fall into the above categories, i.e., patients who did not comply with revisits and those who declined involvement in the study were excluded.
Isolation of S. aureus isolates from high vaginal swabs, oropharynx, breast milk samples and anterior nares of neonates
HVS samples were collected from the posterior fornix from each pregnant patient by the attending physician using sterile bivalve speculum (Changzhou Huankang Medical Devices Co. Ltd, Changzhou City 213116, Jiangsu Province, China) and sterile cotton-tipped applicator (Evepon, Industrial Limited, Onitsha, Anambra State, Nigeria) into freshly prepared sterile thioglycollate medium and incubated at 37°C for 24 h for growth. After growth was observed, a loopful of the sample was streaked initially with the aid of heat- flamed standard aluminium wire loop on to freshly prepared agar media plates – mannitol salt agar (MSA). Thereafter, the plates were incubated aerobically at 37°C for 24 h and examined for growth. Only plates on which colonies appeared were examined. Each distinct colony appearing on agar plates was picked and further studied. Identification was based on cultural and morphologic characteristics such as size, elevation, opacity, and color on media plates. Initial Gram's stain was prepared for each colony, and further identification of each colony was based on their reaction on conventional media. Furthermore, colonies of cocci in clusters resembling staphylococci and fermented mannitol on MSA were presumptively identified as S. aureus and confirmed as such by the coagulase slide and tube agglutination tests with pooled human plasma. Additional confirmatory tests were based on DNase and RNase positivity. Analytical profile index (API) kits of API Staph (bioMéerieux, Marcy l'Etoile France) was also used to autheticate each isolate identity. Similar methods as appropriate were used to identify S. aureus isolates from the oropharynx, breast milk, and anterior nares of neonates.
Antibiotic susceptibility testing
Selected S. aureus isolates were tested for their susceptibility to 23 commonly prescribed antibiotics composed of 15 different classes employing the Kirby-Bauer method. The antibiotics used were obtained from Oxoid (Basingstoke, UK) and included amoxicillin/clavulanic acid AMC (30 μg), ampicillin AMP (10 μg), penicillin G P (1 IU), oxacillin OX (1 μg), ceftriaxone CRO (30 μg), cefuroxime CXM (30 μg), chloramphenicol C (30 μg), imipenem IPM (10 μg), tetracycline TE (30 μg), erythromycin E (15 μg), gentamycin CN (10 μg), kanamycin K (30 μg), streptomycin S (10 μg), vancomycin VA (5 μg), bacitracin BA (10 IU), optochin OPT (5 μg), nalidixic acid NA (30 μg ), ciprofloxacin CIP (5 μg), ofloxacin OFX (5 μg), nitrofurantoin F (300 μg), fusidic acid FD (5 μg), sulphamethoxazole/trimethoprim SXT (25 μg) and mupirocin MUP (200 μg). S. aureus ATCC 25923 and Enterobacter aerogenes ATCC 13042 (American Type Culture Collection, Rockville, USA) were used as control organisms.
Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) of S. aureus strains
The random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used for molecular analysis of the selected bacterial isolates. A modified method of Bitencourt et al., was employed for the DNA extraction.
Random amplified polymorphic DNA-polymerase chain reaction
Ten different random primers each comprising 10 nucleotides in length was employed for the RAPD. The primers and their respective nucleotide sequence is shown in [Table 1]. Fifty nanogram of the DNA template was transferred into a sterile tube and 1 μL of 5 μM of each primer, 1.2 μL of 25 mM MgCl2, 2 μL of 10 mM dNTPs, 1 μL DMSO, 0.2 μL of 2.5 U of Taq DNA polymerase (Promega, USA), 2.5 μL of 10× buffer was added with ultrapure water 15.1 to make a total volume of 25 μL in the tube. The thermocycler was programmed with an initial denaturation at 94°C for 2 min followed by 36 cycles at 94°C for 1 min as the annealing temperature; an additional 1 min at 35°C was followed by extension at 72°C for 3 min and final extension at 72°C for 5 min. The amplicon was stored at 4°C.
|Table 1: Polymerase chain reaction primer sequences used for phylogenic relatedness of S. aureus strains recovered from high vaginal swabs, oropharynx, breast milk, and nares of neonates of HIV seropositive women|
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Agarose gel electrophoresis
PCR products were electrophoresed on 1.5% (W/V) agarose gel in 1× Tis Borate- EDTA (TBE) Buffer at 100 V for 5 h and stained with ethidium bromide (1.0 μg/ml). Thermoscientific O'GeneRuler 100-3000 bp DNA ladder was used as molecular marker to determine the size of each fragments. Gels with amplified bands were visualized and photographed under UV light. Each experiment was tested twice using similar reaction condition at different times for reproducibility of amplified products.
Scoring of random amplified polymorphic DNA gel and formation of data matrices
The random amplified polymorphic DNA gel of each primer was scored based on the appearance of clear or transparent band. The presence of a band was scored as one and absence of a band as zero.
Construction of phylogenetic dendrogram using the dissimilarity analysis and representation for windows
Bacterial strains were analyzed using the Pearson products moment correlation coefficient (r) and the unweighted pair group method with arithmetic averages (UPGMA).
Statistical analysis of data
Statistical evaluation was done using student t-test and one- way analysis of variance (ANOVA) with P ≤ 0.05 as the indicator of statistical significance. SPSS (Chicago, IL, SPSS Inc., 2007) version 17.0 for Windows® was used to perform the analyses.
| Results|| |
[Table 1] depicts the primer sequences and annealing temperature used for random amplified polymorphic DNA polymerase chain reaction. Altogether, 10 primers were employed for the study [Table 1]. [Table 2] also shows the antibiotic resistant pattern of S. aureus recovered from the HVS, oropharynx, breast milk, and anterior nares of neonates of HIV seropositive women. Altogether, 15 phenotypes of S. aureus strains were recovered from the four sites, one of which comprised two strains recovered from the nares of the neonates. Nine of the 16 (56.25%) of the S. aureus isolates tested were MSSA while 7/16 (43.75%) of the isolates were MRSA.
|Table 2: Profile of antibiogram of S. aureus strains recovered from high vaginal swabs, oropharynx, breast milk, and nares of neonates of HIV seropositive women|
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Our data showed that 16 (100%) of the isolates were resistant to nalidixic acid and penicillin, 15/16 (93.75%) were resistant to optochin, and 6/16 (37.5%) to streptomycin [Table 3]. The results also showed that 12/16 (75%) of the strains were resistant to tetracycline, cefuroxime, and nitrofurantoin, 9/16 (56.25%) to chloramphenicol and fusidic acid, and 11/16 (68.75%) to vancomycin. Furthermore, 10/16 (62.5%) of the strains were also resistant to ampicillin, 7/16 (43.75%) to bacitracin and oxacillin, 2/16 (12.5%) to mupirocin and ciprofloxacin, 3/16 (18.75%) to ofloxacin, and 8/16 (50%) each to sulphamethazole/trimethroprim, kanamycin and erythromycin. However, only 1/16 (6.25%) was resistant to imipemen and none to cefriaxone, augumentin, and gentamycin.
|Table 3: Profile of S. aureus distribution among transmitters in relation to age range|
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5] showed the gel electrophoresis of random amplified polymorphic DNA-polymerase chain reaction. Lanes 1, 5, 9, and 13 represent neonates nares; lanes 2, 3, 10, and 11 represent maternal oropharynx; lanes 4, 8, 12, and 16 represent HVS while lanes 6, 7, 14, and 15 represent breast milk samples.
|Figure 1: RAPD-PCR profile of S. aureus isolates recovered from high vaginal swabs, oropharynx, breast milk and nares of neonates. M= marker, lane 1-8 (primer OPB02), Lane 9-16(primer OPH02).|
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|Figure 2: RAPD-PCR profile of S. aureus isolates recovered from high vaginal swabs, oropharynx, breast milk and nares of neonates. |
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|Figure 3: RAPD-PCR profile of S. aureus isolates recovered from high vaginal swabs, oropharynx, breast milk and nares of neonates. M= marker, lane 1-8 (primer OPH09), Lane 9-16(primer OPB12).|
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|Figure 4: RAPD-PCR profile of S. aureus isolates recovered from high vaginal swabs, oropharynx, breast milk and nares of neonates. M= marker, lane 1-8 (primer OPT10), Lane 9-16(primer OPT12).|
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|Figure 5: RAPD-PCR profile of S. aureus isolates recovered from high vaginal swabs, oropharynx, breast milk and nares of neonates. M= marker, lane 1-8 (primer OPT16), Lane 9-16(primer OPT20).|
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| Discussion|| |
This study determined the frequency of colonization and transmission of S. aureus strains from HVS, oropharynx, and breast milk samples among HIV-seropositive pregnant women and the anterior nares of their neonates who attended antenatal clinics in Akure, Ondo State between November 2014 and December 2015. The patients were pregnant women at their third trimester of whom 92.1% were on antiretroviral therapy (ART). Among the 114 HIV-seropositive mothers, altogether, 40/114 (35.08%) were colonized with S. aureus, of which 32/50 (64%) S. aureus isolates were cultured from HVS, 10/50 (20%) from the oropharynx, and 8/50 (16%) from breast milk samples. However, 19/70 (27.14%) of the neonates were colonized with S. aureus. Transmission rate of S. aureus from carrier mothers cultured from the three sites namely HVS, oropharynx, and breast milk was 4/50 (8%). Carrier mother transmission to neonate was 4/19 (21.05%). Our results showed that 16 S. aureus strains were recovered from the patients, of which 7/16(43.75%) was methicillin resistant S. aureus (MRSA) and 9/16 (56.25%) was methicillin sensitive S. aureus (MSSA). Our data also showed that 16 (100%) of the S. aureus strains were resistant to nalidixic acid and penicillin, 15/16 (93.75%) were resistant to optochin and 6/16 (37.5%) to streptomycin. The results also showed that 12/16 (75%) of the strains were resistant to tetracycline and nitrofurantoin, 9/16 (56.25%) to chloramphenicol and fusidic acid, and 11/16 (68.75%) to vancomycin. Furthermore, antibiotic analysis from our data showed 10/16 (62.5%) of the strains were also resistant to ampicillin, 7/16 (43.75%) each to bacitracin and oxacillin, 2/16 (12.5%) each to mupirocin and ciprofloxacin, 3/16 (18.75%) to ofloxacin, and 8/16 (50%) to sulphamethazole/trimethroprim, kanamycin, and erythromycin. Previous studies in this environment have reported the high incidence of resistance of antibiotics, especially with the beta lactams. S. aureus have been known to produce beta lactamases, the enzyme that hydrolytically destroy beta lactams and expression of penicillin- binding protein 2a
(PBP 2a)., It is also interesting to note that, while the majority of the isolates were resistant to the antibiotics employed, only one strain was resistant to imipemen. Studies conducted in this environment have reported high susceptibility of S. aureus isolates to imipemen and gentamycin in immunocompromised individuals and suggested sensitivity to this antbiotic may probably be due to less abuse of the drug, cost, and mode of administration. The genetic relatedness of the four phylogroups among the 16 S. aureus genotypes from carrier mothers showed 81% of S. aureus from maternal oropharynx, 67% of maternal breast milk samples, and 59% from HVS were similar to S. aureus strains recovered from neonatal nares. Furthermore, each maternal-neonate phylogroups was compared; our data showed for maternal-neonate pair A phylogroup, 19/67 (28.35%) from breast milk genotypes, 19/81 (23.45%) from oropharynx, and 26/59 (44.06%) from HVS were genetically related to strains from neonatal nares. Similarly, for maternal-neonate pair B phylogroup, 10/67 (14.92%) from breast milk genotypes, 27/81 (33.33%) from oropharynx, and 7/59 (11.86%) from HVS were genetically related to strains from neonatal nares. Our results also showed that for maternal-neonate pair C phylogroup, 17/67 (25.37%) from breast milk genotypes, 21/81 (25.92%) from oropharynx, and 12/59 (20.33%) from HVS were genetically related to strains from neonatal nares. In addition, maternal-neonate pair D phylogroup, 21/67 (31.34%) from breast milk genotypes, 14/81 (17.28%) from oropharynx, and 14/59 (23.72%) from HVS were genetically related to strains from neonatal nares. Our data showed variance in colonization of S. aureus compared to other studies probably due to the number of mother-neonate pairs and sites of colonization. Mitsuda et al. reported low values compared with our data; 7.5% vaginal colonization rate of S. aureus and 10.1% from the anterior nares. However, their results showed phylogenetic analysis of the samples obatained from the four apparently healthy pregnant carriers, and their infants were completely identical employing pulse-field gel electrophoresis (PFGE).
| Conclusion|| |
In conclusion, our study revealed the frequency of colonization of S. aureus in three sites among HIV seropositive pregnant women and their neonate pairs. HVS displayed highest frequency 64%, 20% oropharynx, and 16% breast milk. Transmission from carrier mothers was 8% which is comparable to other studies. The mode of mother-to-child transmission was also horizontal while phylogroups showed similarities in genetic relatedness. Preponderance of antibiotic resistance was evident among the genotypes from mother-to-child, which is epidemiological important in the propensity of neonates to acquire resistant organisms early in life.
The authors acknowledge the Doctors, Dr. Akintan Adesina, Dr. A K Aderoba, nursing staff, and laboratory technologists at the four healthcare centres.
Financial support and sponsorship
Conflicts of Interest
The authors declare no conflicts of interest.
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Kwashie Ajibade Ako-Nai
Department of Microbiology, Faculty of Science, Obafemi Awolowo University, Ile Ife, Osun State
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]