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\begin{document}


\def\RunningAuthor{Samira Fattah Hamid and Aza Bahadeen Taha}
\firstPage{6095}
\articleType{Original Article}
\receivedDate{27 Apr 2020}
\acceptedDate{01 Jun 2020}
\revisedDate{27 May 2020}
\journalVolume{2020, 11}
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\journalDoi{ijrps.v11i4.3281}
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\title{\textbf{Mannitol Fermenting Methicillin-Resistant Coagulase Negative Staphylococci Isolated From Diabetic Foot Infections}}
\author{Samira~Fattah~Hamid\textsuperscript{*}\textsuperscript{1}, 
            Aza~Bahadeen~Taha\textsuperscript{2}~\\[5pt]{\textsuperscript{1}Department of Basic Sciences, College of Medicine, Hawler Medical University, Erbil, Kurdistan region-Iraq}~\\{\textsuperscript{2}Department of Nursing, College of Nursing, Hawler Medical University, Erbil, Kurdistan region-Iraq}}

\begin{abstract}
Detection of mannitol fermenting coagulase-negative staphylococci is frequently unnoticed when \textit{Staphylococcus} \textit{aureus} is screening in the laboratory. On the other hand, the emergence of coagulase-negative staphylococci as critical human pathogens need  dependable methods for the identification of clinically significant coagulase-negative staphylococci to understand  the epidemiology of infections caused by these bacteria. The study aimed to identify mannitol fermenting coagulase-negative staphylococci that assumed to be \textit{Staphylococcus aureus} as they formed  yellow colonies on Mannitol Salt agar plates. Samples were taken from eighty-four patients with diabetic foot infections. The specimen was cultured on Blood agar and Mannitol Salt agar. Mannitol fermenting coagulase-negative staphylococci isolates diagnosed through Vitek2 system then confirmed by detecting \textit{16S rRNA } gene and absence of\textit{ the nuc} gene. Antibiotic sensitivity and methicillin resistance were detected by Vitek2 system, then methicillin resistance was confirmed by Oxacillin Salt Agar Screen test and detection of \textit{the mecA } gene. Out of 81 \textit{Staphylococcus } isolated from foot and nose of diabetic foot patients, twenty isolates were mannitol fermenting coagulase-negative staphylococci, they related to following species; \textit{Staphylococcus haemolyticus}, \textit{staphylococcus lentus},\textit{ Staphylococcus xylosus, Staphylococcus lugdunensis, Staphylococcus hominis, Staphylococcus galinarum } and\textit{ Staphylococcus saprophyticus}). The majority of them (85\%) were phenotypically methicillin-resistant and genotypically harbouring \textit{mecA } gene. 80\% were resistant to Erythromycin, 70\% to Clindamycin, 35\% to Trimethoprim-Sulphamethoxazole, 30\% to Gentamicin and Rifampicin, 15\% to Levofloxacin and Teicoplanin. 30\% expressed inducible clindamycin resistance.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}coagulase-negative,\newline staphylococcus,\newline diabetic foot,\newline mannitol
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Samira~Fattah~Hamid~\\ Phone:\ ~\\  Email:\ samira.hamid@hmu.edu.krd
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
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\section{Introduction}
\textit{Staphylococcus} \textit{aureus } (\textit{ S}. \textit{aureus}) is the primary  etiological agent of skin and nares, \unskip~\citep{877542:20256834,877542:20256836}, and most common causative agent in diabetic foot infections \unskip~\citep{877542:20256830}. Due to its clinical importance, a large  number of diagnostic tests are used  to identify \textit{S}. \textit{aureus};\textit{\space } growth on Mannitol Salt Agar plate (MSA) is the most widely used. MSA plate was developed in the year 1945 to selectively isolate  pathogenic staphylococci in one  step \unskip~\citep{877542:20256841}. MSA is a selective medium containing  7{\textendash}9 \% Nacl which allows \textit{S}. \textit{aureus} to grow and produce yellow colonies with yellow zones due to fermentation of mannitol sugar which in turn lead to  drop in the medium's pH. In contrast, coagulase-negative staphylococci (CONS) produce pink to red colonies and no change in the medium colour \unskip~\citep{877542:20256834,877542:20256837}.

There are some  reports about  CONS that they also can ferment mannitol  and produce yellow colonies on MSA \unskip~\citep{877542:20256842,877542:20256840}. Mannitol Salt positive CONS interrupt the isolation and then  identification of \textit{S}. \textit{aureus} on the primary plate. Unfortunately, false-positive results caused by  coagulase-negative staphylococci can lead to  overestimation of \textit{S}. \textit{aureus} infection rates \unskip~\citep{877542:20256848}. 

Coagulase-negative staphylococci considered as one of the  most frequently isolated pathogens in a clinical laboratory. They are now gradually becoming  an essential causative agent in various infections like wounds \unskip~\citep{877542:20256840,877542:20256850}. They are increasingly recognised as pathogens in case of diabetic foot wound infections \unskip~\citep{877542:20256849}. CONS acquired  multiple antibiotic-resistance mechanisms, especially  methicillin resistance \unskip~\citep{877542:20256846}. The problem is worsened by the fact that methicillin-resistant staphylococci, in addition to beta-lactam antibiotics, are resistant to other antibiotic classes  including macrolides, aminoglycosides, fluoroquinolones and tetracyclines \unskip~\citep{877542:20256852}. There is a believed that CONS might act as a significant reservoir  for antibiotic resistance genes, which might  be transferred among \textit{Staphylococcus} genus \unskip~\citep{877542:20256853,877542:20256831}. Therefore, accurate identification of CONS species is of diagnostic value and clinical importance. 

This study aimed to identify these CONS that isolated from diabetic foot infections and nares of diabetic patients, which assumed to be \textit{S. aureus} as they form yellow colonies on MSA plates.
    
\section{Materials and Methods}
\textbf{Study place and period}

From December 2016 to January 2018 eighty-four patients with diabetic foot ulcer who hospitalised in Rizgari and Hawler teaching hospitals in Erbil city/ Iraq were included in this study.

\textbf{Ethical approval}

The ethics committee of the College of Medicine/ Hawler Medical University approved this study before conducting the study (paper code: 15- date: 23/4/2016). Informed consent was collected from patients before obtaining Specimens. 

\textbf{Inclusion criteria}

The patient had diabetes mellitus with foot ulcers, accidentally diagnosed as diabetic Mellitus after admission with a foot ulcer, patient with gangrene of the foot complicated by diabetes.

\textbf{Exclusion criteria}

Patient not willing to participate, had foot infections but were not diabetic, had gangrene of the foot. Still, the aetiology wasn't due to diabetes complication, had a healed ulcer site, pregnant or under 18 years old.

\textbf{Collection and culture of specimens}

 Specimens were collected aseptically from infected foot lesions and nose of diabetic patients by using a sterile cotton swab \unskip~\citep{877542:20256832} and cultured on Blood agar and Mannitol Salt agar plates (Lab M / UK), then incubated at 37\ensuremath{^\circ}C and checked after 24 - 48 hours. 


\bgroup
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\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/a3daeb71-87c0-4146-b231-7811e0789f79-upicture1.png}{\includegraphics{images/a3daeb71-87c0-4146-b231-7811e0789f79-upicture1.png}}{}
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\caption{\boldmath {Mannitol fermenting CON Staphylococcus haemolyticus on Mannitol Salt agar media}}
\label{f-0a2c5c9d472c}
\end{figure}
\egroup

\begin{table*}[!htbp]
\caption{\boldmath {Primers to detect CONS of bacteria identity genes and methicillin-resistant genes} }
\label{tw-f4ef821effd1}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2079\linewidth-2\tabcolsep}p{\dimexpr.4411\linewidth-2\tabcolsep}p{\dimexpr.10939999999999998\linewidth-2\tabcolsep}p{\dimexpr.2416\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Gene name & Nucleotide sequence & Bp & References \\
\tblmidrule 
16S rRNA-F \mbox{}\protect\newline 16S rRNA-R &
  5'-GTA GGT GGG AAG CGT TAT CC - 3'  \mbox{}\protect\newline 5'-CGC ACA TCA GCG TCA G - 3.'  &
  218 &
  \unskip~\citep{877542:20256854}\\
nuc-F \mbox{}\protect\newline nuc-R &
  5'-GCG ATT GAT GGT GAT ACG GTT {\textendash} 3'  \mbox{}\protect\newline 5'-AGC CAA GCC TTG ACG AAC TAA AGC - 3'  &
  280 &
  \unskip~\citep{877542:20256854}\\
mecA-F \mbox{}\protect\newline mecA-R &
  5'-CTC AGG TAC TGC TAT CCA CC- 3'  \mbox{}\protect\newline 5'-CAC TTG GTA TAT CTT CAC C - 3'  &
  448 &
  \unskip~\citep{877542:20256851}\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Mannitol-fermenting CONS identity confirmed by Vitek2 system} }
\label{tw-bf51768e32ca}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.24759999999999998\linewidth-2\tabcolsep}p{\dimexpr.49290000000000006\linewidth-2\tabcolsep}p{\dimexpr.2595\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}No  &  Bacteria & No. of isolates\\
\tblmidrule 
1 &
  \textit{Staphylococcus haemolyticus} &
  7\\
2 &
  \textit{Staphylococcus lentus} &
  6\\
3 &
  \textit{Staphylococcus xylosus} &
  2\\
4 &
  \textit{Staphylococcus lugdunensis} &
  2\\
5 &
  \textit{Staphylococcus hominis} &
  1\\
6 &
  \textit{Staphylococcus galinarum} &
  1\\
7 &
  \textit{Staphylococcus saprophyticus} &
  1\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Mannitol-fermenting CONS identity confirmed by \textit{16S} \textit{rRNA} and \textit{nuc} genes} }
\label{tw-85b8f8a44034}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0936\linewidth-2\tabcolsep}p{\dimexpr.09870000000000001\linewidth-2\tabcolsep}p{\dimexpr.21240000000000002\linewidth-2\tabcolsep}p{\dimexpr.1765\linewidth-2\tabcolsep}p{\dimexpr.1894\linewidth-2\tabcolsep}p{\dimexpr.2294\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}No. & Patient code & CONS of bacteria  & Location of isolation  & \textit{16S rRNA gene} & \textit{Nuc gene}\\
\tblmidrule 
1 &
  5 &
  \textit{S. haemolyticus} &
  Nose  &
  Positive  &
  Negative \\
2 &
  25 &
  \textit{S. haemolyticus} &
  Left Foot &
  Positive &
  Negative\\
3 &
  34 &
  \textit{S. haemolyticus} &
  Nose  &
  Positive &
  Negative\\
4 &
  63 &
  \textit{S. haemolyticus} &
  Nose  &
  Positive &
  Negative\\
5 &
  71 &
  \textit{S. haemolyticus} &
  Nose  &
  Positive &
  Negative\\
6 &
  71 &
  \textit{S. haemolyticus} &
  Right Foot &
  Positive &
  Negative\\
7 &
  84 &
  \textit{S. haemolyticus} &
  Left Foot &
  Positive &
  Negative\\
8 &
  19 &
  \textit{S. lentus} &
  Nose  &
  Positive &
  Negative\\
9 &
  34 &
  \textit{S. lentus} &
  Right foot &
  Positive &
  Negative\\
10 &
  42 &
  \textit{S. lentus} &
  Left Foot &
  Positive &
  Negative\\
11 &
  43 &
  \textit{S. lentus} &
  Left Foot &
  Positive &
  Negative\\
12 &
  73 &
  \textit{S. lentus} &
  Nose  &
  Positive &
  Negative\\
13 &
  73 &
  \textit{S. lentus} &
  Left Foot &
  Positive &
  Negative\\
14 &
  6 &
  \textit{S. xylosus} &
  Right Foot &
  Positive &
  Negative\\
15 &
  55 &
  \textit{S. xylosus} &
  Left Foot &
  Positive &
  Negative\\
16 &
  47 &
  \textit{S. lugdunensis} &
  Nose  &
  Positive &
  Negative\\
17 &
  50 &
  \textit{S. lugdunensis} &
  Nose  &
  Positive &
  Negative\\
18 &
  14 &
  \textit{S. hominis} &
  Left Foot &
  Positive &
  Negative\\
19 &
  77 &
  \textit{S. galinarum} &
  Left Foot &
  Positive &
  Negative\\
20 &
  84 &
  \textit{S. saprophyticus} &
  Right Foot &
  Positive &
  Negative\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Confirmation of mannitol fermenting CONS resistance to methicillin by phenotype and genotype methods} }
\label{tw-427bed7686db}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.0834\linewidth-2\tabcolsep}p{\dimexpr.14719999999999999\linewidth-2\tabcolsep}p{\dimexpr.2188\linewidth-2\tabcolsep}p{\dimexpr.14079999999999998\linewidth-2\tabcolsep}p{\dimexpr.2184\linewidth-2\tabcolsep}p{\dimexpr.1914\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor} &  &  & \multicolumn{2}{p{\dimexpr(.3592\linewidth-2\tabcolsep)}}{\cAlignHack Methicillin resistance test results} & \\
\rowcolor{kwdboxcolor}No. & Patient code & Bacteria & Vitek2 system & Oxacillin Salt-Agar Screen Test  & mecA gene \\
\tblmidrule 
1 &
  5 &
  \textit{S. haemolyticus} &
  Positive  &
  Negative  &
  Positive\\
2 &
  25 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
3 &
  34 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
4 &
  63 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
5 &
  71 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
6 &
  71 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
7 &
  84 &
  \textit{S. haemolyticus} &
  Positive &
  Positive &
  Positive\\
8 &
  19 &
  \textit{S. lentus} &
  Positive &
  Positive &
  Positive\\
9 &
  34 &
  \textit{S. lentus} &
  Positive &
  Positive &
  Positive\\
10 &
  42 &
  \textit{S. lentus} &
  Positive &
  Positive  &
  Positive\\
11 &
  43 &
  \textit{S. lentus} &
  Negative  &
  Positive &
  Positive\\
12 &
  73 &
  \textit{S. lentus} &
  Positive &
  Positive &
  Positive\\
13 &
  73 &
  \textit{S. lentus} &
  Positive &
  Positive &
  Positive\\
14 &
  6 &
  \textit{S. xylosus} &
  Negative  &
  Negative  &
  Positive\\
15 &
  55 &
  \textit{S. xylosus} &
  Negative  &
  Negative  &
  Positive\\
16 &
  47 &
  \textit{S. lugdunensis} &
  Positive &
  Positive &
  Positive\\
17 &
  50 &
  \textit{S. lugdunensis} &
  Positive &
  Positive &
  Positive\\
18 &
  14 &
  \textit{S. hominis} &
  Positive &
  Positive &
  Positive\\
19 &
  77 &
  \textit{S. galinarum} &
  No* &
  Positive &
  Positive\\
20 &
  84 &
  \textit{S. saprophyticus} &
  No* &
  Positive &
  Positive\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{No*= No Vitek data are given}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Antibiotic resistance patterns of mannitol fermenting CONS} }
\label{tw-f023680a810c}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.13310000000000002\linewidth-2\tabcolsep}p{\dimexpr.17729999999999997\linewidth-2\tabcolsep}p{\dimexpr.2748\linewidth-2\tabcolsep}p{\dimexpr.41480000000000004\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}No. & Patient code & CONS of bacteria  & Resistance to antibiotics \\
\tblmidrule 
1 &
  5 &
  \textit{S. haemolyticus } &
  Lev,  Ery, Tet, TMP-SMX\\
2 &
  25 &
  \textit{S. haemolyticus} &
  Ery, Clin, Tet, \\
3 &
  34 &
  \textit{S. haemolyticus} &
  Teic, Tet, TMP-SMX\\
4 &
  63 &
  \textit{S. haemolyticus} &
  No resistance to any tested antibiotics\\
5 &
  71 &
  \textit{S. haemolyticus} &
  Gen, Ery, Clin, Rif\\
6 &
  71 &
  \textit{S. haemolyticus} &
  Gen, Ery, Clin, Rif\\
7 &
  84 &
  \textit{S. haemolyticus} &
  Lev, Ery, Tet, TMP-SMX\\
8 &
  19 &
  \textit{S. lentus} &
  Gen, Tob, Ery, Clin, Tet, Rif\\
9 &
  34 &
  \textit{S. lentus} &
  Gen, Tob, Lev, Ery, Clin, Teic, Rif, TMP-SMX\\
10 &
  42 &
  \textit{S. lentus} &
  Ery, Clin, Tet, Rif\\
11 &
  43 &
  \textit{S. lentus} &
  Ery, Clin., Tet\\
12 &
  73 &
  \textit{S. lentus} &
  Ery, Clin, Tet\\
13 &
  73 &
  \textit{S. lentus} &
  Ery, Clin, Tet\\
14 &
  6 &
  \textit{S. xylosus} &
  Ery, Clin, Tet, TMP-SMX\\
15 &
  55 &
  \textit{S. xylosus} &
  Clin, Tet\\
16 &
  47 &
  \textit{S. lugdunensis} &
  Ery, Clin, Tet, Rif\\
17 &
  50 &
  \textit{S. lugdunensis} &
  Ery, Clin, Tet\\
18 &
  14 &
  \textit{S. hominis} &
  Gen, Tob, Ery, Clin, Teic, Tet, TMP-SMX\\
19 &
  77 &
  \textit{S. galinarum} &
  No Vitek data\\
20 &
  84 &
  \textit{S. saprophyticus} &
  Gen, Ery, Tet, TMP-SMX\\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{Lev=Levofloxacin,Ery=Erythromycin, Tet=tetracycline, TMP-SMX= Trimethoprim/Sulfamethoxazole, Clin=Clindamycin, Teic=Teicoplanine, Gen=Gentamicin,Rif=Rifampicin, Tob=Tobramycin.}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{Presumptive identification of bacteria}

After growth, if bacteria in the culture media were a mix, they purified by streak plate method to create a pure culture of one bacteria and determine the phenotypic character of the bacteria like mannitol fermentation and yellow pigment production.

\textbf{Identification by Vitek2 system }

The Vitek2 system (BioM{\'e}rieux Vitek, France) was used in this study to confirm the identification of all isolated bacteria.

\textbf{Antibiotic susceptibility testing by Vitek2 system}

Susceptibility test card "AST-GP580 TEST KIT- REF.22233" for Gram-positive was used to perform susceptibility testing for different antibiotics according to the manufacturer's instructions.

\textbf{Methicillin resistance detection in isolated bacteria}

\textbf{1. Vitek2 system}

Detection of Cefoxitin and Oxacillin resistance investigated with the same card used in the antibiotic susceptibility testing, as mentioned above.

\textbf{2. Oxacillin Salt Agar Screen Test}

Mueller-Hinton agar (Lab M / UK) plates containing 4\% NaCl and six \ensuremath{\mu}g/ml of Oxacillin powder were prepared and inoculated with 1 \ensuremath{\mu}l of 0.5 McFarland suspension of the bacterium by streaking in one quadrant and incubated at 33 to 35\ensuremath{^\circ} C for 24 hrs. (because incubating above 35\ensuremath{^\circ}C may not detect methicillin-resistant strains). Plates were scrutinised with transmitted light for {\textgreater} 1 colony or light film of growth ({\textgreater} 1 colony = oxacillin resistant) \unskip~\citep{877542:20256845}.

\textbf{Extraction of genomic DNA}

Genomic DNA extracted by using EzWay\textsuperscript{TM} Genomic DNA Kit, Bacterial (KOMABIOTECH/South Korea) following the manufacturer's instructions.

\textbf{Genotype identification of \textit{Staphylococci } isolates\textit{\space } and detection of \textit{mecA} gene}

Primers for the \textit{16S rRNA} gene was used to confirm genus, and species-specific \textit{nuc} gene to confirm \textit{aureus } species \unskip~\citep{877542:20256840}. Also, methicillin resistance was confirmed by using a primer for detection of \textit{mecA} gene as illustrated in Table~\ref{tw-f4ef821effd1}.

\textbf{Data analysis} \textbf{\space }

Data were analysed using SPSS version 23.0 software (SPSS, Inc., Chicago, IL, USA).
    
\section{Results}
The samples were taken from eighty-four diabetic foot patients who participate in this study. Mean age of the patients was 57.21\ensuremath{\pm}11.64 years. Males represented 54.8\%, while females were 45.2\%.

Out of 81 staphylococci strains, 20 coagulase-negative staphylococci\textit{} were able to ferment\textit{\space } mannitol sugar on Mannitol Salt agar plates, as shown in Table~\ref{tw-bf51768e32ca}  and Figure~\ref{f-0a2c5c9d472c}. The Vitek2 system determined the identity of these isolates. Confirmation made through detecting \textit{16S} \textit{rRNA} gene to confirm them as \textit{Staphylococcus} genus and through searching for \textit{nuc} gene (absence of \textit{nuc} gene confirm phenotypic identification of these isolates), all isolates were positive for \textit{16S} \textit{rRNA} and negative for \textit{nuc} gene as shown in Table~\ref{tw-85b8f8a44034}.

Majority of these bacteria were resistant to methicillin phenotypically by Vitek2 system method (83.3\%) and Oxacillin Salt Agar Screen Test (85\%). All of them (100\%) found to harbour \textit{mecA} gene as seen in Table~\ref{tw-427bed7686db}.

Sixteen (80\%) out of 20 Mannitol fermenting CONS were resistant to Erythromycin, 14 (70\%) to Clindamycin, 7 (35\%) to Trimethoprim-Sulphamethoxazole, while the resistance to Gentamicin and Rifampicin was 6(30\%), to Levofloxacin and Teicoplanin was 3(15\%). Six (30\%) of the isolates expressed inducible clindamycin resistance. None of the isolates was resistant to Moxifloxacin, Linezolid, Vancomycin and Tigecycline. 85\% of the isolates were multidrug-resistant (MDR) as they showed  resistance to at least three classes of antibiotics (Table~\ref{tw-f023680a810c}).
    
\section{Discussion}
Some microbiologists often depend  on differential  test and growth on selective media to diagnose bacteria, which may  lead to wrong identification of some bacteria and result in the wrong prescription of antibiotics in which may later lead to therapeutic failure. Production  of yellow colonies due to fermentation of Mannitol sugar is considered  as a presumptive tool to identify \textit{S.} \textit{aureus} and to differentiate it  from coagulase-negative staphylococci. Still, some researchers reported that some of the CONS also could  produce yellow colonies on MSA \unskip~\citep{877542:20256852,877542:20256840}. Repeatedly isolation of CONS from patients with infection should be taken seriously as important human pathogens, dependable methods for proper diagnosis and treatment of CONS are such essential to understand the epidemiology of infections caused by these bacteria \unskip~\citep{877542:20256833}.

Contrary to reports that mentioned MSA as a useful tool in which it can be used to  discriminate between \textit{S}. \textit{aureus} and CONS. This study provides the first report on different species of CONS that can ferment mannitol as \textit{S}. \textit{haemolyticus}, \textit{S. lentus}, \textit{S. xylosus}, \textit{S. lugdunensis}, \textit{S. hominis}, \textit{S. galinarum } and\textit{ S. saprophyticus } that isolated from diabetic foot infections and grown on MSA with characteristic production of yellow colonies. The Vitek2 system confirmed them, \textit{16S}\textit{rRNA} gene and nuc gene, The absence of the \textit{S. aureus} species-specific nuc gene confirmed  phenotypic identification as CONS \unskip~\citep{877542:20256840}. \unskip~\citet{877542:20256843} misreported the accuracy of Vitek2 system identifying CONS isolates. \unskip~\citet{877542:20256848} reported 7\% false-positive results and 31\% false-negative results  when colonies are identified  by colour alone on differential culture media. These isolates were also confirmed phenotypically and genotypically as methicillin-resistant. Same results were published by \unskip~\citet{877542:20256852,877542:20256841,877542:20256840}. \unskip~\citet{877542:20256839} mentioned that methicillin-resistant CONS are the predominant cause of nosocomial infections, which greatly limit therapeutic options for opportunistic infections and may play a role in spreading resistances within the community  \unskip~\citep{877542:20256838,877542:20256847}. Also \unskip~\citet{877542:20256835} suggests that a negative methicillin-resistant nares swab is useful to predict the absence of methicillin resistance in a subsequent culture from a diabetic foot infection because guidelines recommend empiric methicillin resistance coverage in patients who have had methicillin resistance previously. There is toxicity related to antibiotics for empiric methicillin resistance coverage, especially vancomycin, and for that reason they recommend methicillin resistance \textit{Staphylococcus} \textit{aureus} nares screening at admission to hospital. Besides resistance to beta-lactam antibiotics, the methicillin-resistant CONS in this study also showed  resistance to other antibiotic classes like  macrolides, lincosamides, sulfonamides, aminoglycosides and rifamycins. murugesan et al. reported a similar  finding. For CONS isolated from nares of hemodialysis patients \unskip~\citep{877542:20256844}. Although the degree of importance of multidrug-resistant methicillin-resistant CONS is not clearly understood, they may serve as  a reservoir for resistance genes. They may spread from them  to pathogenic bacteria within and across species and genera \unskip~\citep{877542:20256852}.

This finding indicates that a single phenotypic test cannot provide dependable  results in the identification and differentiation of \textit{S. aureus } with CONS, and a combination of tests should be used to avoid misidentification  of isolates.
    
\section{Conclusions}
This study, along with earlier investigations, indicates that there is the high false identification of \textit{S. aureus} and other  tests are needed to differentiate between \textit{S}. \textit{aureus} and CONS colonies that ferment mannitol  on MSA. So educating microbiologist working in clinical laboratories about this misidentification and recommending using more than one confirmatory test is so important to decrease the rate of false-positive identification. 
\section*{Acknowledgement}We thank all participants and the nurses who assisted in sample collection.

\textbf{Conflicts of interest}

The authors declare that there is no conflict of interest regarding the publication of this paper.

\textbf{Funding support}

This study formed part of a PhD program (No.6220-1-1), funded by Hawler Medical University-Iraq.


    

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