Molecular Study of Vancomycin Resistance in Hospital Acquired Staphylococcus Infection

Purpose: Vancomycin is treatment of choice in MRSA infection. In the last years there is increasing incidence of vancomycin resistance either vancomycin intermediate S. aureus (VISA) or vancomycin-resistant staphylococci (VRSA). This increasing in resistance strains is challenging problem especially in hospital acquired infection. The present study was carried out to find the incidence of(VISA)and(VRSA) among MRSA in children hospital in Egypt with correlation of molecular study for mec A gene and Vancomycin resistance gene Van A, Van B and Van C genes by multiplex PCR. Method: Out of 678 hospital acquired infection (365) staphylococcal isolates was obtained in which coagulase negative were (152) (41.6%) and positive (213) (58.4%) were isolated from various samples which confirmed to be hospital acquired infection according to CDC guidelines. All Staphylococcal aureus isolates and coagulase negative (CoNS) were suspected to antibiotic disc diffusion. MRSA strain were subjected to MIC for vancomycine and molecular study for detection mec A and Van A, Van B and Van C genes by multiplex PCR Results: MRSA was reported in (103) staphylococcal and (10) CONS mec gene was present in all MRSA stain. Vancomycine resistance gene was not found in any of CoNS. While in staphylococcal aureus vanA was present in 2 VRSA and 4 VISA isolates and van B was present in 6 VISA and in one VRSA isolates vanC gene was not found either in Staphylococcus aureus nor CoNS. Conclusion: The results of the current study illustrate the emergence of vancomycin resistance among methicillin-resistant S. aureus isolated from children with healthcare-associated infections. The majority revealed the occurrence of vanA and vanB as an accountable mechanism for this resistance. Citation: Zaki MES, Abouelnour A, El-Kannishy SMH, Hassan R (2019) Molecular Study of Vancomycin Resistance in Hospital Acquired Staphylococcus Infection. Int J Drug Dev & Res 11: 11-14 Volume 11(3): 11-14 (2019)-012 Int J Drug Dev & Res ISSN: 0975-9344 Minimal Inhibitory Concentrations (MICs) for vancomycin The determination of minimal inhibitory concentrations (MICs) for vancomycin was performed by the use of standardized agar dilution techniques. Briefly, gradient plates of Mueller-Hinton agar of vancomycin (0.5–128 μg/ml). With direct colony suspension method 0.5 McFarland equivalent inoculums were prepared in normal saline from 18–24 h agar plate culture. The suspension then was diluted to achieve needed inoculum concentration. All strains were placed into gradient plates. Plates were incubated overnight at 35°C for any visible growth. Readings were taken according to NCCLS guidelines [17-20]. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 strains were used as vancomycin sceptible controls and E. faecalis ATCC 51299 as vancomycin resistant control. Vancomycin resistance among MRSA according to MIC was classified into susceptible, intermediate susceptible and resistant according to CLSI, 2009 [10]. Detection of mecA gene by PCR


Introduction
Staphylococcus is a major pathogen in hospital acquired infections. It has variety of virulence factors and capable of acquiring resistance to most of antibiotics making S. aureus a "superbug. In the last decade, methicillin resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide [1]. MRSA is difficult in treatment because it is resistant to methicillin as well as to other β-lactam antibiotic, with the exception of glycopeptides antibiotics [2,3] Vancomycin as glycopeptides \have been used as a therapeutic alternative for the treatment of MRSA [4], but due its excessive use resistant strains have been reported to reemerge among S. aureus species. The mechanism of vancomycin resistance in S. aureus is not fully known. It is may be due to association of Vancomycin-resistant genes with S. aureus species. These genes are seven types of resistance genes namely (van A, B, C, D, E, G, and L) which are usually transferred by transposon Tn1546 [5].
The aim of the present study was to identify the emergence of vancomycin-resistant S. aureus among MRSA resistant strains and to identify the occurrence of vanA, vanB and van C genes among resistant isolates.

Isolates
The study was conducted at Mansoura University Children hospital, Egypt from December 2014 till March 2017. The study included isolated S. aureus strains from children diagnosed to have health care associated infections (HCAI) according to CDC [4]. The patients signed written consents and the study was approved by Mansoura Faculty of Medicine ethical committee. The strains were from variety of hospital acquired infection including pus blood sputum catheters swabs and urine CULRUER Blood agar and Macconkey agar were used for samples except for urine samples which were inoculated on CLED the samples were incubated for 24-48h at 37C.

Identification of Staphylococci
The included samples were from various clinical samples such as blood, pus and body fluids. The isolates were identified as S. aureus by standard biochemical techniques and conventional methods (colony morphology, Gram stain, catalase activity, and slide and tube coagulase test) after primary culture of the clinical samples.

Minimal Inhibitory Concentrations (MICs) for vancomycin
The determination of minimal inhibitory concentrations (MICs) for vancomycin was performed by the use of standardized agar dilution techniques. Briefly, gradient plates of Mueller-Hinton agar of vancomycin (0.5-128 μg/ml). With direct colony suspension method 0.5 McFarland equivalent inoculums were prepared in normal saline from 18-24 h agar plate culture. The suspension then was diluted to achieve needed inoculum concentration. All strains were placed into gradient plates. Plates were incubated overnight at 35°C for any visible growth. Readings were taken according to NCCLS guidelines [17][18][19][20]

DNA preparation
One colony of pure culture was suspended in 25 µL of sterile water and the suspension was put in the water bath at 100°C for 12 minutes. One micron of the suspension was used for PCR amplification. The oligonucleotide primers for mecA gene (mecA F 5'GTAGAAATGACTGAACGTCCGATGA 3' and mecA R 5CCAATTCCACATTGTTTCGGTCTAA 3') A Biometra DNA thermocycler was programmed with the initial denaturation,4 min at 94°C; 30 cycles with a 45-s denaturation step at 94°C, a 45-s annealing step at 56°C and a 30-s extension step at 72°C and 2 min extension step at 72°Cand a holding step at 4°C until the sample was analyzed. After amplification 10 µL of the reaction mixture was loaded into a 1% agarose gel stained with 10 µL ethidium bromide and electrophoreses to estimate the sizes of the amplification products with a 100-bp molecular size standard ladder (Sigma). The positive tests have shown PCR product of 310 bp. Sterile distilled water was used as a negative control under complete sterile standard precautions for PCR. S. aureus ATCC 29213 reference strain was used as mecA positive control strain.

Multiplex PCR for Van A, B, C genes DNA preparation
One colony of pure culture was suspended in 25 µL of sterile water and the suspension was put in the water bath at 100°C for 12 minutes. One micron of the suspension was used for PCR amplification [17].

Discussion
Hospital acquired infection caused by resistance stains represents real challenge to heath care system [1]. This study reported the presence of MRSA in (30.9%) among HAIs in children hospital during the period of the study. Where Staphylococcus aureus (28.2%) and CoNS was (2.7%). Similar rates were reported in studies done in developing countries [12], while lower rates were reported in developed countries such as USA [21,22].
Our findings demonstrated high resistance of MRSA to mostly common used beta-lactams and macrolides antibiotics with rates from 85% up to 100%. These high rates of resistance are also reported from other studies [14,15]. The high rate of resistance could be explained that in developing countries like ours there is irrational use of antibiotic due to its availability at the drug store without prescription also uncontrolled use in hospitals and uncontrolled use in agriculture, animal industry and fisheries [5], As the infection by MRSA consider a strong risk factor to vancomycin resistance and vancomycin resistance was not reported from methicillin-susceptible strain [22]. This goes with our pattern of MIC to vancomycin as there is resistance to vancomycin only in MRSA and not in MISSA strain. Also our result shows that VISA pattern was present in two CONS stain and thirteen staph. Auras, and VRSA pattern was present in five isolates of Staphylococcus aureus not present in CONS Van C --- vancomycin 8 μg/ml was isolated from urine specimen of patient (10years old) who was admitted in renal ward, and the other with vancomycin 4μg/ml was isolated blood of patient (6years old) who was admitted to intensive care unit. The vancomycin resistance in CoNS either VRSA or VISA have been reported in other hospital as in Brazil which reported vancomycin resistant strains of CoNS [9]. And in India also reported the emergence of vancomycin resistant in CoNS [11].
Our result shows that highest resistance to vancomycin was reported in thirteen years old girl with renal failure isolates no [p11] with MIC of vancomycin is 64 µg/ml with recurrent hospital admission with last one was more than 10 days with history of vancomycin use and it was positive to van A gene and mec gene.
All VRSA isolates had mec gene and the patient had history of recurrent hospital admission with last one was more than five days with history of vancomycin use. The data of the VISA and VRSA is shown in Table 2 in relation to the detection of mec gene and van gene and clinical history.
The mechanism of transmission of hospital acquired resistance strain could be by many ways from patients or health care worker as well as from the environmental surfaces, while the transmission of coagulase-negative staphylococci is not fully known but it's probably the same rout of transmission [20]. Also the duration of hospital stay and history of glycopeptides antibiotics and to some extent the causes of underlying diseases as renal failure appear to be significant risk factor in increasing the resistance [22].
Vancomycin resistance in S. aureus mechanism is not known it would be that S. aureus acquire (Van gene) vancomycin resistance gene [15]. The result of multiplex pcr in this study shows that vancomycin resistance gene were not present in all isolates. But the absence of vanA/B genes in the isolates dose not rule out that these strains are not VRSA or VISA as there is another hypothesis which stat that cell wall thickening is play role in vancomycin resistance. Biochemical and transmission electron microscopy (TEM) examination of the Mu50 cell, show that increased of peptidoglycan. More murein monomers and more layers (probably 30-40 layers as judged by cell-wall thickness observed with TEM) of peptidoglycan are to be present in the cell wall. As a result, more vancomycin molecules are trapped in the peptidoglycan layers before reaching the cytoplasmic membrane where peptidoglycan synthesis occurs [21].
The widespread of vancomycin resistance in hospitals lead to focusing on infection control role in emerging of vancomycin-resistant staphylococci. In our study the Hospital Infection Control Practices Advisory Committee (HICPAC) of the infection control guidelines for staphylococci resistance was followed To detect how it was implemented and its role in spreading the resistance strains. In Table  3 how much implantation of infection control practices was checked in two CONS with intermediates resistance to vancomycin and in Staphylococcus aureus with VRSA. It was reported that not all the items were implemented in our hospital this strong risk factor to increases the rate of resistance isolates.

Conclusion
Our study shows the magnitude of VRSA or VISA in hospital acquired infection and a strict regulation on use of antibiotics with implementation of infection control policy is the effective way that we could do to prevent the spread of the resistance strain.

Funding
This work was not supported by any financial funding.

Conflict of Interest
The authors declare that they have no conflict of interest.

Ethical Approval
All procedures performed in this study involving human participants