# First Record of Isolation and Characterization of Methicillin ResistantStaphylococcus lugdunensis from Clinical Samples in Iraq

Authors: Alaa H. Al-Charrakh; Mohammed H. Obayes
Journal: BioMed Research International
 (2014)

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## Abstract

This study was conducted to determine the frequency ofStaphylococcus lugdunensis in different clinical samples. Out of 690 clinical samples, a total of 178 coagulase negative staphylococci (CoNS) isolates were recovered. CoNS were identified as 10 different species; 22 isolates belonged to Staphylococcus lugdunensis. Two specific genes for S. lugdunensiswere used ( tanAgene and fblgene) to confirm identification. Both of these specific genes were detected in 15 (68.1%) of 22 isolates that were identified phenotypically. The results of oxacillin MIC showed that 7 of the 15 (46.6%) S. lugdunensisisolates were oxacillin resistant. The antibiotic susceptibility testing against 16 antibiotics showed that resistance rates were variable towards these antibiotics. Eight of fifteen S. lugdunensisisolates (53.3%) were β-lactamase producer. Results of molecular detection of mecAgene found that mecAgene was detected in 6 (40%) of 15 S. lugdunensis. All of these 6 isolates (S1, S2, S3, S4, S5, and S6) were resistant to oxacillin. One isolate (S7) was resistant to oxacillin but mecAwas not detected in this isolate. This study is a first record of isolation and characterization of methicillin resistant S. lugdunensis(MRSL) from clinical samples in Iraq.


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## Body

## 1. Introduction

The coagulase-negative staphylococci (CoNS) is a large group of Gram-positive bacteria most often found colonizing the skin and mucosal surfaces of humans and other mammals [1]. Several species of CoNS are recognized as potential pathogens, mainly causing nosocomial infections, often involved in infections related to implanted medical devices such as intravenous catheters, prosthetic heart valves, and orthopedic implants.Staphylococcus lugdunensisis a coagulase-negative staphylococci first described by Freney and his colleagues in 1988 [2]. The organism is found as a skin commensal in healthy individuals.S. lugdunensis has been implicated in invasive diseases, especially fulminant native and prosthetic-valve endocarditis [3]. Other invasive infections include brain abscess, meningitis, skin abscesses, soft tissue infections, spondylodiscitis, foreign body infections, and peritonitis [4].S. lugdunensis shares a number of potential virulence factors withS. aureus. In particular,S. lugdunensis may express a clumping factor and produce a thermostable DNase [5].S. lugdunensis produces a tannase (tannin acyl hydrolase) that degrades hydrolysable tannins [6]. The phenotypic biological tests, such as the ornithine decarboxylase test and genotypic molecular tests, have been developed to identify these bacteria [7]. Several nucleic acid targets that permit the differentiation ofS. lugdunensis from other CoNS have been exploited using molecular methods. These include the 16S rRNA gene, which was used to confirm identity ofS. lugdunensis isolate [8], and therpoB gene, which is also specific toS. lugdunensis [4]. ThetanA gene that coded tannase acyl hydrolase was detected inS. lugdunensis [6]. A fibrinogen-binding protein known as Fbl that encoded byfbl gen is specific toS. lugdunensis [9]. ThemecA gene has been reported in several data, the first in a neonate [10] with methicillin resistantS. lugdunensis (MRSL) that produces an alternative penicillin binding protein (PBP2A). Although several researchers have been reported phenotypic and molecular characterizations of methicillin resistantS. lugdunensis (MRSL) worldwide, no information are available about these resistant bacteria in Iraq. Therefore the main goals of this study were to isolate and determine the antibiotic resistance patterns of these important bacteria from clinical samples and detect the presence ofmecA gene that encodes methicillin resistance to confirm being MRSL.


## 2. Materials and Methods

### 2.1. Patients and Samples Collection

This study included 630 patients (aged 2 days–70 years) suffering from different infections who were admitted to four health centers in Al-Hilla city, Iraq. These patients were admitted to different hospital wards, in addition to swabs taken from private clinics during a period extending from November 2012 to the end of May 2013.Swabs of different samples 690 were generally collected from different sites (wound, burn, blood culture, subaxillary, urine, stool, sputum, throat, ear, skin lesion, high vaginal, and other different swabs). Each sample was immediately inoculated on the blood agar plates and mannitol salt agar. The swab has been inoculated on culture media and incubated aerobically for 24 hours at 37°C. Information about age, antibiotic usage, residence, and hospitalization of patients was taken into consideration.


### 2.2. Bacterial Isolates

Staphylococcus lugdunensis isolates were recovered and identified based on their morphology, Gram-staining, catalase test, coagulase test, and ornithine decarboxylation test [11]. Identification was confirmed using two specific genes (tanA andfbl genes) by PCR assay [12].


### 2.3. Screening ofβ-Lactam Resistant Isolates

FifteenS. lugdunensis isolated were subjected to β-lactam resistance screening test as a phenotypic selection test. Preliminary screening ofS. lugdunensis isolates resistance to β-lactam antibiotics was carried out by using pick and patch method on Muller-Hinton agar plates supplemented with ampicillin. All of 15S. lugdunensis isolates were subjected to oxacillin resistance screening test by using the same method on Muller-Hinton agar plates supplemented with 4% NaCl and oxacillin 6 μg/mL [13].


### 2.4. Antimicrobial Susceptibility Testing

The antimicrobial susceptibility patterns of isolates to different antibiotics were determined using disk diffusion test and interpreted according to CLSI guidelines [13]. The following antibiotics were obtained (from Oxoid, UK, and Himedia, India) as standard reference disks as known potency for laboratory use: ampicillin (10 μg), oxacillin (5 μg), cloxacillin (5 μg), cefoxitin (30 μg), amoxicillin-clavulanate (20/10 μg), cefexime (30 μg), ceftriaxone (30 μg), imipenem (10 μg), azithromycin (15 μg), and doxycycline (30 μg). The susceptibility to ampicillin, oxacillin, and vancomycin was also determined using twofold agar dilution method [14].


### 2.5. Detection ofβ-Lactamase Production

Nitrocefin diagnostic disk (Fluka, Switzerland) was used to detect the ability of 15 isolates to produceβ-lactamase. A number of required nitrocefin disks were placed into sterile empty Petri dish and moistened with one drop of sterile D.W.; then the disk was holed by sterile forceps and wiped across a young colony on agar plate. The development of a red color in the area of the disk where the culture was applied indicated a positive result.


### 2.6. Detection oftanA,fbl, andmecA Genes

Three genes were detected in present study, firsttanA gene that coded to tannase acyl hydrolase enzyme that degrades tannin. The second gene wasfbl gene that coded to fibrinogen binding protein. The third gene that was detected in the present study wasmecA that was responsible for oxacillin/methicillin resistance by coding for penicillin binding protein (PBP2a); the primer sequence of these genes were (tanA F: AGCATGGGCAATAACAGCAGTAA,tanA R: GCTGCGCCAATTTGTTCTAAATAT) 239 bp; the conditions were 95°C 3 min 1x, 94°C 20 sec, 60°C 20 sec 25x, 72°C, 20 sec, and 72°C 5 min 1x [12].For (fbl F: GTAAATAGCGAGGCACAAGC,fbl R: GGTAAATCGTATCTGCCGCT) 425 bp, the conditions were 94°C 3 min 1x, 94°C 1 min, 60°C 1 min 30x, 72°C 1 min, and 72°C 5 min 1x [15]. For (mecA F: TCCAGGAATGCAGAAAGACCAAAGC,mecA R: GACACGATAGCCATCTTCATGTTGG) 499 bp, the conditions were 94°C 3 min 1x, 94°C 1.5 min, 55°C 1 min 36x, 72°C 1 min, and with final step 72°C 10 min 1x [16].


### 2.7. Bacteriological Analysis and Antibiotic Susceptibility of“S. pseudolugdunensis” Isolates

“Staphylococcus pseudolugdunensis” isolates recovered in this study were identified using the same methods as inS. lugdunensis. According to [17], any isolate that diagnosed phenotypically asS. lugdunensis but it was negative totanA andfbl genes was rediagnosed as“S. pseudolugdunensis.” The antibiogram, β-lactamase production, and presence ofmecA gene for these isolates were also determined as above.


## 2.1. Patients and Samples Collection

This study included 630 patients (aged 2 days–70 years) suffering from different infections who were admitted to four health centers in Al-Hilla city, Iraq. These patients were admitted to different hospital wards, in addition to swabs taken from private clinics during a period extending from November 2012 to the end of May 2013.Swabs of different samples 690 were generally collected from different sites (wound, burn, blood culture, subaxillary, urine, stool, sputum, throat, ear, skin lesion, high vaginal, and other different swabs). Each sample was immediately inoculated on the blood agar plates and mannitol salt agar. The swab has been inoculated on culture media and incubated aerobically for 24 hours at 37°C. Information about age, antibiotic usage, residence, and hospitalization of patients was taken into consideration.


## 2.2. Bacterial Isolates

Staphylococcus lugdunensis isolates were recovered and identified based on their morphology, Gram-staining, catalase test, coagulase test, and ornithine decarboxylation test [11]. Identification was confirmed using two specific genes (tanA andfbl genes) by PCR assay [12].


## 2.3. Screening ofβ-Lactam Resistant Isolates

FifteenS. lugdunensis isolated were subjected to β-lactam resistance screening test as a phenotypic selection test. Preliminary screening ofS. lugdunensis isolates resistance to β-lactam antibiotics was carried out by using pick and patch method on Muller-Hinton agar plates supplemented with ampicillin. All of 15S. lugdunensis isolates were subjected to oxacillin resistance screening test by using the same method on Muller-Hinton agar plates supplemented with 4% NaCl and oxacillin 6 μg/mL [13].


## 2.4. Antimicrobial Susceptibility Testing

The antimicrobial susceptibility patterns of isolates to different antibiotics were determined using disk diffusion test and interpreted according to CLSI guidelines [13]. The following antibiotics were obtained (from Oxoid, UK, and Himedia, India) as standard reference disks as known potency for laboratory use: ampicillin (10 μg), oxacillin (5 μg), cloxacillin (5 μg), cefoxitin (30 μg), amoxicillin-clavulanate (20/10 μg), cefexime (30 μg), ceftriaxone (30 μg), imipenem (10 μg), azithromycin (15 μg), and doxycycline (30 μg). The susceptibility to ampicillin, oxacillin, and vancomycin was also determined using twofold agar dilution method [14].


## 2.5. Detection ofβ-Lactamase Production

Nitrocefin diagnostic disk (Fluka, Switzerland) was used to detect the ability of 15 isolates to produceβ-lactamase. A number of required nitrocefin disks were placed into sterile empty Petri dish and moistened with one drop of sterile D.W.; then the disk was holed by sterile forceps and wiped across a young colony on agar plate. The development of a red color in the area of the disk where the culture was applied indicated a positive result.


## 2.6. Detection oftanA,fbl, andmecA Genes

Three genes were detected in present study, firsttanA gene that coded to tannase acyl hydrolase enzyme that degrades tannin. The second gene wasfbl gene that coded to fibrinogen binding protein. The third gene that was detected in the present study wasmecA that was responsible for oxacillin/methicillin resistance by coding for penicillin binding protein (PBP2a); the primer sequence of these genes were (tanA F: AGCATGGGCAATAACAGCAGTAA,tanA R: GCTGCGCCAATTTGTTCTAAATAT) 239 bp; the conditions were 95°C 3 min 1x, 94°C 20 sec, 60°C 20 sec 25x, 72°C, 20 sec, and 72°C 5 min 1x [12].For (fbl F: GTAAATAGCGAGGCACAAGC,fbl R: GGTAAATCGTATCTGCCGCT) 425 bp, the conditions were 94°C 3 min 1x, 94°C 1 min, 60°C 1 min 30x, 72°C 1 min, and 72°C 5 min 1x [15]. For (mecA F: TCCAGGAATGCAGAAAGACCAAAGC,mecA R: GACACGATAGCCATCTTCATGTTGG) 499 bp, the conditions were 94°C 3 min 1x, 94°C 1.5 min, 55°C 1 min 36x, 72°C 1 min, and with final step 72°C 10 min 1x [16].


## 2.7. Bacteriological Analysis and Antibiotic Susceptibility of“S. pseudolugdunensis” Isolates

“Staphylococcus pseudolugdunensis” isolates recovered in this study were identified using the same methods as inS. lugdunensis. According to [17], any isolate that diagnosed phenotypically asS. lugdunensis but it was negative totanA andfbl genes was rediagnosed as“S. pseudolugdunensis.” The antibiogram, β-lactamase production, and presence ofmecA gene for these isolates were also determined as above.


## 3. Results and Discussion

### 3.1. Isolation and Identification ofS. lugdunensis Isolates

A total of 690 clinical samples were collected; 602 (87.24%) gave positive growth on blood agar medium, while 88 (12.76%) gave no growth. Out of 393 Gram-positive bacteria, 306 (77.8%) were identified as staphylococci based on morphological characteristics and biochemical tests. According to result of coagulase test, the 306 staphylococci isolates were divided into coagulase positive 128 (41.8%) and coagulase negative 178 (58.2%) (Table1).Table 1
Numbers, sources, and percentages of coagulase negative staphylococci isolates.
Source
Number (%)
CoNS No. (%)
Wound swabs
105 (17.4)
42 (23.56)
Ear swabs
28 (4.6)
6 (3.37)
Urine swabs
50 (8.3)
11 (6.17)
Skin lesion swabs
25 (4.1)
10 ( 5.6)
Throat swabs
32 (5.3)
11 (6.17)
Burn swabs
80 (13.2)
15 (8.4)
Blood culture
69 (11.4)
13 (7.3)
Sputum
36 (5.9)
4 (2.24)
Sub axillary swabs*
64 (10.6)
52 (29.2)
HVS**
23 (8.3)
2 (1.12)
Stool swabs
38 (6.3)
2 (1.12)
Other
52 (8.6)
10 ( 5.6)
Total
602 (100)
178 (100)
*These samples were taken from immature patients in neonate intensive care unit.
*
*High vaginal swabs.Result of present study was similar to that of Bouza and his colleagues [18], who found that bacterial isolates from clinical samples included 70.7% Gram-positive, 22.2% of Gram-negative, and 7.2% of yeast; they also found thatS. aureus and CoNS constituted 40% and 60%, respectively. In a local study in Iraq, Al-Fuadi [19] found that total of 148 bacterial isolates represented by different Gram-positive and Gram-negative bacteria in percentage of 77% and 23% respectively. He also found that a total of 31 of 100Staphylococcus isolates belonged toS. aureus. This difference may have belonged to variation of samples collected in this study.Results also showed that the highest percentages of CoNS in subaxillary swabs and wound swabs were 29.2% and 23.5%, respectively. The prevalence ofS. lugdunensis was 22 (12.3%), which is higher than results of several researchers. This may be due to the fact that depending on phenotypic characteristics alone is insufficient and may result in misidentification ofS. lugdunensis. So, the present study depended (in addition to phenotypic characteristics) on the genotypic characteristics (PCR) to confirm the result. Depending on PCR results, out of 22 of CoNS that were identified phenotypically asS. lugdunensis isolates, 15 (8%) were identified asS. lugdunensis, while the other seven isolates were belonged to “S. pseudolugdunensis.”Clinical isolates were as follows: subauxiliary swab (4), skin swabs (2), wound (3), burn (1), blood (1), throat swab (2), ear swab (1), and peritonitis (1), while noS. lugdunensis isolates were recovered from urine, sputum swabs, stool swabs, and high vaginal swabs. Skin swabs represented folliculitis, boils, and abscesses. Koksal and his colleagues [20] found that it constituted 9% of CoNS isolates from blood culture, while other researchers found thatS. lugdunensis constituted only 3.3% of CoNS collected from different samples [21].Results revealed that the seven“S. pseudolugdunensis” isolates were recovered from clinical samples of skin (1), urine (1), burn swabs (2), subauxiliary swabs (2), and acne (1).


### 3.2. Molecular Characterization ofS. lugdunensis Isolates

Definite phenotypic identification of a Gram-positive, catalase-positive coccus asS. lugdunensisimplies a negative tube coagulase test and positive ornithine decarboxylase activities [22]. However, complete hemolytic, yellow pigmentation, and detection of a fibrinogen affinity factor, although not consistently expressed byS. lugdunensis,may leadto its misidentification asS. aureus[1].S. lugdunensisis an unusually virulent coagulase-negative species, associated with severe infection. So, using single-step, species-specific PCR protocol forS. lugdunensisidentification is very important [15].


#### 3.2.1. Detection oftanAGene

The specifictanAgene forS. lugdunensiswas detected in 15 (68.1%) of 22 isolates that were identified phenotypically. These 15 isolates were identified asS. lugdunensis(Figure 1). The remaining 7 isolates (31.9%) were reidentified as“S. pseudolugdunensis” [17]. Result also found thatS. aureusandS. epidermidisthat were used as negative control had notanA gene which confirms the result of Noguchi and his coworkers [12] who found that no gene or protein homologous totanA were found in a similarity search using published databases such as Gen Bank. These results strongly suggest thattanAis specific toS. lugdunensis.Gel electrophoresis of PCR oftanA amplicon (239 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14: number ofS. lugdunensis isolates from different clinical samples.
(a)
(b)


#### 3.2.2. Detection offblGene ofS. lugdunensisIsolates

A suitable nucleic acid target to diagnosedS. lugdunensisisfblgene, which encods a fibrinogen-binding adhesin [15]. The gene was detected in all 15S. lugdunensis isolates that were positive totanAin this study (Figure 2), while no amplification product was obtained fromS. aureusandS. epidermidisisolates that were used as negative control as in Figure 3.Figure 2
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, and 7: number ofS. lugdunensis isolates from different clinical samples.Figure 3
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, and 15: number ofS. lugdunensis isolates. C1:S. aureus, C2:S. epidermidis.According to results of PCR results, among 22S. lugdunensisthat diagnosed phenotypically, 15 isolates were found to be positive totanAandfblgenes that were specific toS. lugdunensis [4], so other isolates (number = 7) were diagnosed as“S. pseudolugdunensis” [17].


### 3.3. Primary Screening ofβ-Lactam Resistant Isolates

The results of screening test showed that 11 isolates (73.3%) ofS. lugdunensiswere resistant to ampicillin. All these isolates were able to grow normally in the presence of ampicillin; this may be attributed to most ofS. lugdunensis isolates (about 90% of them) that are coming from several infectious sources (nosocomial infections and other anatomical sites) that are resistant to penicillin [23] due to production of β-lactamases that acts in the hydrolysis of β-lactam ring of penicillin which is transformed into acid neutralizing its bactericidal effect [23].The results of oxacillin resistant screening test showed that 7 of the 11 (63.6%)β-lactam resistantS. lugdunensisisolates were oxacillin resistant. This result was in concordance with study of Mateo and her coworkers [24] who referred to identifying methicillin resistance using the Vitek 2 system and the Wider system, and they found that 47.1% of strains were considered resistant to methicillin.


### 3.4. Susceptibility toβ-Lactam Antibiotics

The results revealed that 11 of 15S. lugdunensisisolates showed high resistance (73.3%) to ampicillin (Figure 4). Results also showed that the resistance rate to oxacillin and cloxacillin was 46.6%. Methicillin replaces methicillin as oxacillin which is stable under storage conditions, and methicillin actually is an excellent inducer of themecA gene. Ezekiel and Adebayo [25] isolated three strains ofS. lugdunensisof 149 CoNS; all isolates were resistant to oxacillin and other β-lactam antibiotics. Tan and his colleagues [22] in Singapore found that resistance to oxacillin was detected in 5% of the isolates.Figure 4
Percentages of antibiotic resistance amongStaphylococcus lugdunensis isolates. Amp: ampicillin; Clx: cloxacillin; Oxn: oxacillin; Cfx: cefoxitin; Cft: ceftriaxone; Cfz-Clv: ceftazidime clavulunate; Cfm: cefixime; Ipm: imipenem; Amv: amoxiclav; Azn: azithromycin; Akn: amikacin; Cln: clindamycin; Rfn: rifampicin; Dox: doxycycline; Vcn: vancomycin; TMP-SMZ: trimethoprim/sulfamethoxazole.Results of cefoxitin (2nd generation), ceftriaxone, and cefexime (3rd generation) showed that the percentages ofS. lugdunensisresistant isolates were substantial to these antibiotics: 46.6%, 53.3%, and 40%, respectively (Figure 4). These results can be explained by the fact that all staphylococcal strains produce β-lactamase which destroys the β-lactam ring resulting in inactive products [26]. Tan and his colleagues [22] found that resistance to cefoxitin was detected in 5% of isolates.The resistance rates to amoxiclav and ceftazidime-clavulanic acid were 60% and 53.3%, respectively. Clavulanic acid can inhibit the action ofβ-lactamase enzyme that causes decrease in the resistance of bacteria to β-lactam antibiotics [27]. Results found thatS. lugdunensisisolates were susceptible to imipenem (80%). Imipenem inhibits bacterial cell wall synthesis by binding to and inactivating PBPs [28].


### 3.5. Susceptibility to Non-β-Lactam Antibiotics

Result of this study regarding susceptibility to amikacin found that the isolates showed low level of resistance (46.6%) to this antibiotic. The resistance rate of azithromycin was 53% (Figure4). This resistance may be attributed to the efflux mechanism in staphylococci which is mediated byMsrA, a protein that induced by clarithromycin, azithromycin, and telithromycin and encoded bymsrA gene [29]. Result of this study regarding susceptibility to clindamycin found that the isolates showed low level of resistance (46.6%) to this antibiotic. Hellbacher et al. [30] found 10% ofS. lugdunensis isolates were resistant to clindamycin.Staphylococcus lugdunensisisolates results showed (73%) resistance to doxycycline. Tan and his colleagues [22] in Singapore found that resistance to tetracycline was 12% of isolates. The percentage of resistance for both trimethoprim-sulfamethoxazole and rifampin was 66%. Rifampin resistance inEscherichia coliandS. aureusis due to alterations in the target leading to a reduced affinity of the enzyme for the antibiotic [31].


### 3.6. Results of Antibiotic Resistance by MIC

In this study, 11 of 15 (73.3%)S. lugdunensisisolates were resistant to ampicillin (≥128 μg/mL), while 4 of 15 were having MIC values reached 2 μg/mL. The MIC values ofS. lugdunensisisolates against oxacillin revealed that 5 of 15 isolates reached 32 μg/mL, while MIC value of 2 isolates was ≥ 64 μg/mL. Six oxacillin resistant isolates (S1, S2, S3, S4, S5, and S6) hadmecAgene, but one isolate (S7) did not have such gene. Out of 15S. lugdunensisisolates (detected by MIC method), 14 isolates (93.2%) were sensitive to vancomycin, while only one isolate (6.8%) showed reduced susceptibility to vancomycin 8 μg/mL (intermediate resistant). Bourgeois and his coworkers [31] found that 6/13S. lugdunensisisolates were tolerant to vancomycin. No isolates showed any degree of resistance to vancomycin as in many data. ThevanA genes that are responsible for resistance (vangenes) are inducible and transferable and confer high-level resistance to vancomycin [32].


### 3.7.β-Lactamase Production

Eight isolates (53.3%) wereβ-lactamase producer. All these isolates were ampicillin resistant; seven of eight β-lactamase producing isolates were oxacillin resistant, while the remaining one was oxacillin sensitive. Six of eight were havingmecAgene (Table 2).Table 2
Relationship between ampicillin, oxacillin resistance with presence ofmecAgene, and β-lactamase production in Staphylococcus lugdunensisisolates.
Isolate number
Ampicillin resistant
Oxacillin resistant
mecA
β-Lactamase production
S1
+
+
+
+
S2
+
+
+
+
S3
+
+
+
+
S4
+
+
+
+
S5
+
+
+
+
S6
+
+
+
+
S7
+
+
−
+
S8
+
−
−
−
S9
+
−
−
−
S10
+
−
−
−
S11
+
−
−
+
S12
−
−
−
−
S13
−
−
−
−
S14
−
−
−
−
S15
−
−
−
−Mateo and her coworkers [24] found that 11.8% ofS. lugdunensiswere β-lactamase producers. Several authors reported that the percentages of β-lactamase-positiveS. lugdunensis vary from 24 to 40% in US isolates collections [33]. Papapetropoulos and his colleagues [34] isolated 14S. lugdunensisstrains from clinical specimens (abscesses and wounds) of 250 bed-hospital in Athens, Greece; 5 (30.2%) ofS. lugdunensiswere β-lactamase positive. The difference between this study and other studies may be due to the fact that the global using of β-lactam antibiotics in Iraq may result in induction of bacterial resistance to β-lactams via production of β-lactamases [35].


### 3.8. Detection ofmecAGene in MRSL Isolates

In this studymecAgene was detected in 6 (40%) of 15S. lugdunensis(Figure 5). All of these 6 isolates (S1, S2, S3, S4, S5, and S6) were resistant to oxacillin (Table 2). One isolate (S7) was resistant to oxacillin, butmecAwas not detected in this isolates.Gel electrophoresis of PCR ofmecA amplicon (499 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, and 6:mecA positive; Lanes S7, 8, 9, 10, 11, 12, 13, and 14:mecA negative samples.
(a)
(b)Staphylococcus lugdunensisis generally considered to be susceptible to oxacillin. Several studies reported negative PCR results when screening formecA, but among reports in the English literaturemecAhas been detected in twoS. lugdunensisisolates [8, 36]. Tee and his colleagues [8] reported a case of MRSL causing bloodstream infection in a neonate with an oxacillin MIC > 256 mg/L havingmecAgene. In 2008, Tan and his colleagues [22] found five (4.7%)S. lugdunensisstrains carrying themecAgene in a collection of 106 clinical isolates.


### 3.9. Antibiotic Susceptibility and Detection ofmecA Gene in“S. pseudolugdunensis” Isolates

The antibiotic susceptibility profile of“S. pseudolugdunensis” isolates (number = 7) is shown in Table 3. In addition to the results discussed earlier, this study represents the first record of characterization of oxacillin resistant andmecA positive“S. pseudolugdunensis” isolates recovered from clinical specimens in Iraqi hospitals (Table 4).Table 3
Antibiotic susceptibility profile of“S. pseudolugdunensis” isolates.
Isolate number
Ceftazidime-clavulanate
Ceftriaxone
Azithromycin
Imipenem
Doxycycline
amikacin
Cefixim
Amoxiclav
Vancomycin
Clindamycin
Rifampicin
TMP-SMZ
SP1
S
S
S
S
R
R
R
S
S
R
S
R
SP2
S
S
S
S
R
S
R
S
S
S
S
S
SP3
R
R
R
S
R
R
R
S
S
R
R
R
SP4
S
S
S
S
R
S
S
R
S
S
S
R
SP5
R
R
S
R
R
S
S
S
S
S
S
R
SP6
S
R
S
S
S
S
R
S
S
R
S
S
SP7
S
S
S
S
S
R
R
R
S
S
S
STable 4
Relationship between oxacillin resistance with presence ofmecA gene and β-lactamase production in “S. pseudolugdunensis” isolates.
Isolate number
Resistance to
β-Lactamase production
mecAgene
Ampicillin
Oxacillin 4μg/ml
Oxacillin  6μg/ml
Cefoxitin
SP1
+
+
+
−
+
−
SP2
+
+
+
+
+
+
SP3
+
+
+
+
+
+
SP4
+
+
+
+
+
+
SP5
+
+
−
−
−
−
SP6
+
+
+
+
+
+
SP7
+
−
−
−
−
−


## 3.1. Isolation and Identification ofS. lugdunensis Isolates

A total of 690 clinical samples were collected; 602 (87.24%) gave positive growth on blood agar medium, while 88 (12.76%) gave no growth. Out of 393 Gram-positive bacteria, 306 (77.8%) were identified as staphylococci based on morphological characteristics and biochemical tests. According to result of coagulase test, the 306 staphylococci isolates were divided into coagulase positive 128 (41.8%) and coagulase negative 178 (58.2%) (Table1).Table 1
Numbers, sources, and percentages of coagulase negative staphylococci isolates.
Source
Number (%)
CoNS No. (%)
Wound swabs
105 (17.4)
42 (23.56)
Ear swabs
28 (4.6)
6 (3.37)
Urine swabs
50 (8.3)
11 (6.17)
Skin lesion swabs
25 (4.1)
10 ( 5.6)
Throat swabs
32 (5.3)
11 (6.17)
Burn swabs
80 (13.2)
15 (8.4)
Blood culture
69 (11.4)
13 (7.3)
Sputum
36 (5.9)
4 (2.24)
Sub axillary swabs*
64 (10.6)
52 (29.2)
HVS**
23 (8.3)
2 (1.12)
Stool swabs
38 (6.3)
2 (1.12)
Other
52 (8.6)
10 ( 5.6)
Total
602 (100)
178 (100)
*These samples were taken from immature patients in neonate intensive care unit.
*
*High vaginal swabs.Result of present study was similar to that of Bouza and his colleagues [18], who found that bacterial isolates from clinical samples included 70.7% Gram-positive, 22.2% of Gram-negative, and 7.2% of yeast; they also found thatS. aureus and CoNS constituted 40% and 60%, respectively. In a local study in Iraq, Al-Fuadi [19] found that total of 148 bacterial isolates represented by different Gram-positive and Gram-negative bacteria in percentage of 77% and 23% respectively. He also found that a total of 31 of 100Staphylococcus isolates belonged toS. aureus. This difference may have belonged to variation of samples collected in this study.Results also showed that the highest percentages of CoNS in subaxillary swabs and wound swabs were 29.2% and 23.5%, respectively. The prevalence ofS. lugdunensis was 22 (12.3%), which is higher than results of several researchers. This may be due to the fact that depending on phenotypic characteristics alone is insufficient and may result in misidentification ofS. lugdunensis. So, the present study depended (in addition to phenotypic characteristics) on the genotypic characteristics (PCR) to confirm the result. Depending on PCR results, out of 22 of CoNS that were identified phenotypically asS. lugdunensis isolates, 15 (8%) were identified asS. lugdunensis, while the other seven isolates were belonged to “S. pseudolugdunensis.”Clinical isolates were as follows: subauxiliary swab (4), skin swabs (2), wound (3), burn (1), blood (1), throat swab (2), ear swab (1), and peritonitis (1), while noS. lugdunensis isolates were recovered from urine, sputum swabs, stool swabs, and high vaginal swabs. Skin swabs represented folliculitis, boils, and abscesses. Koksal and his colleagues [20] found that it constituted 9% of CoNS isolates from blood culture, while other researchers found thatS. lugdunensis constituted only 3.3% of CoNS collected from different samples [21].Results revealed that the seven“S. pseudolugdunensis” isolates were recovered from clinical samples of skin (1), urine (1), burn swabs (2), subauxiliary swabs (2), and acne (1).


## 3.2. Molecular Characterization ofS. lugdunensis Isolates

Definite phenotypic identification of a Gram-positive, catalase-positive coccus asS. lugdunensisimplies a negative tube coagulase test and positive ornithine decarboxylase activities [22]. However, complete hemolytic, yellow pigmentation, and detection of a fibrinogen affinity factor, although not consistently expressed byS. lugdunensis,may leadto its misidentification asS. aureus[1].S. lugdunensisis an unusually virulent coagulase-negative species, associated with severe infection. So, using single-step, species-specific PCR protocol forS. lugdunensisidentification is very important [15].


### 3.2.1. Detection oftanAGene

The specifictanAgene forS. lugdunensiswas detected in 15 (68.1%) of 22 isolates that were identified phenotypically. These 15 isolates were identified asS. lugdunensis(Figure 1). The remaining 7 isolates (31.9%) were reidentified as“S. pseudolugdunensis” [17]. Result also found thatS. aureusandS. epidermidisthat were used as negative control had notanA gene which confirms the result of Noguchi and his coworkers [12] who found that no gene or protein homologous totanA were found in a similarity search using published databases such as Gen Bank. These results strongly suggest thattanAis specific toS. lugdunensis.Gel electrophoresis of PCR oftanA amplicon (239 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14: number ofS. lugdunensis isolates from different clinical samples.
(a)
(b)


### 3.2.2. Detection offblGene ofS. lugdunensisIsolates

A suitable nucleic acid target to diagnosedS. lugdunensisisfblgene, which encods a fibrinogen-binding adhesin [15]. The gene was detected in all 15S. lugdunensis isolates that were positive totanAin this study (Figure 2), while no amplification product was obtained fromS. aureusandS. epidermidisisolates that were used as negative control as in Figure 3.Figure 2
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, and 7: number ofS. lugdunensis isolates from different clinical samples.Figure 3
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, and 15: number ofS. lugdunensis isolates. C1:S. aureus, C2:S. epidermidis.According to results of PCR results, among 22S. lugdunensisthat diagnosed phenotypically, 15 isolates were found to be positive totanAandfblgenes that were specific toS. lugdunensis [4], so other isolates (number = 7) were diagnosed as“S. pseudolugdunensis” [17].


## 3.2.1. Detection oftanAGene

The specifictanAgene forS. lugdunensiswas detected in 15 (68.1%) of 22 isolates that were identified phenotypically. These 15 isolates were identified asS. lugdunensis(Figure 1). The remaining 7 isolates (31.9%) were reidentified as“S. pseudolugdunensis” [17]. Result also found thatS. aureusandS. epidermidisthat were used as negative control had notanA gene which confirms the result of Noguchi and his coworkers [12] who found that no gene or protein homologous totanA were found in a similarity search using published databases such as Gen Bank. These results strongly suggest thattanAis specific toS. lugdunensis.Gel electrophoresis of PCR oftanA amplicon (239 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14: number ofS. lugdunensis isolates from different clinical samples.
(a)
(b)


## 3.2.2. Detection offblGene ofS. lugdunensisIsolates

A suitable nucleic acid target to diagnosedS. lugdunensisisfblgene, which encods a fibrinogen-binding adhesin [15]. The gene was detected in all 15S. lugdunensis isolates that were positive totanAin this study (Figure 2), while no amplification product was obtained fromS. aureusandS. epidermidisisolates that were used as negative control as in Figure 3.Figure 2
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, 6, and 7: number ofS. lugdunensis isolates from different clinical samples.Figure 3
Gel electrophoresis of PCR offbl amplicon (425 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, and 15: number ofS. lugdunensis isolates. C1:S. aureus, C2:S. epidermidis.According to results of PCR results, among 22S. lugdunensisthat diagnosed phenotypically, 15 isolates were found to be positive totanAandfblgenes that were specific toS. lugdunensis [4], so other isolates (number = 7) were diagnosed as“S. pseudolugdunensis” [17].


## 3.3. Primary Screening ofβ-Lactam Resistant Isolates

The results of screening test showed that 11 isolates (73.3%) ofS. lugdunensiswere resistant to ampicillin. All these isolates were able to grow normally in the presence of ampicillin; this may be attributed to most ofS. lugdunensis isolates (about 90% of them) that are coming from several infectious sources (nosocomial infections and other anatomical sites) that are resistant to penicillin [23] due to production of β-lactamases that acts in the hydrolysis of β-lactam ring of penicillin which is transformed into acid neutralizing its bactericidal effect [23].The results of oxacillin resistant screening test showed that 7 of the 11 (63.6%)β-lactam resistantS. lugdunensisisolates were oxacillin resistant. This result was in concordance with study of Mateo and her coworkers [24] who referred to identifying methicillin resistance using the Vitek 2 system and the Wider system, and they found that 47.1% of strains were considered resistant to methicillin.


## 3.4. Susceptibility toβ-Lactam Antibiotics

The results revealed that 11 of 15S. lugdunensisisolates showed high resistance (73.3%) to ampicillin (Figure 4). Results also showed that the resistance rate to oxacillin and cloxacillin was 46.6%. Methicillin replaces methicillin as oxacillin which is stable under storage conditions, and methicillin actually is an excellent inducer of themecA gene. Ezekiel and Adebayo [25] isolated three strains ofS. lugdunensisof 149 CoNS; all isolates were resistant to oxacillin and other β-lactam antibiotics. Tan and his colleagues [22] in Singapore found that resistance to oxacillin was detected in 5% of the isolates.Figure 4
Percentages of antibiotic resistance amongStaphylococcus lugdunensis isolates. Amp: ampicillin; Clx: cloxacillin; Oxn: oxacillin; Cfx: cefoxitin; Cft: ceftriaxone; Cfz-Clv: ceftazidime clavulunate; Cfm: cefixime; Ipm: imipenem; Amv: amoxiclav; Azn: azithromycin; Akn: amikacin; Cln: clindamycin; Rfn: rifampicin; Dox: doxycycline; Vcn: vancomycin; TMP-SMZ: trimethoprim/sulfamethoxazole.Results of cefoxitin (2nd generation), ceftriaxone, and cefexime (3rd generation) showed that the percentages ofS. lugdunensisresistant isolates were substantial to these antibiotics: 46.6%, 53.3%, and 40%, respectively (Figure 4). These results can be explained by the fact that all staphylococcal strains produce β-lactamase which destroys the β-lactam ring resulting in inactive products [26]. Tan and his colleagues [22] found that resistance to cefoxitin was detected in 5% of isolates.The resistance rates to amoxiclav and ceftazidime-clavulanic acid were 60% and 53.3%, respectively. Clavulanic acid can inhibit the action ofβ-lactamase enzyme that causes decrease in the resistance of bacteria to β-lactam antibiotics [27]. Results found thatS. lugdunensisisolates were susceptible to imipenem (80%). Imipenem inhibits bacterial cell wall synthesis by binding to and inactivating PBPs [28].


## 3.5. Susceptibility to Non-β-Lactam Antibiotics

Result of this study regarding susceptibility to amikacin found that the isolates showed low level of resistance (46.6%) to this antibiotic. The resistance rate of azithromycin was 53% (Figure4). This resistance may be attributed to the efflux mechanism in staphylococci which is mediated byMsrA, a protein that induced by clarithromycin, azithromycin, and telithromycin and encoded bymsrA gene [29]. Result of this study regarding susceptibility to clindamycin found that the isolates showed low level of resistance (46.6%) to this antibiotic. Hellbacher et al. [30] found 10% ofS. lugdunensis isolates were resistant to clindamycin.Staphylococcus lugdunensisisolates results showed (73%) resistance to doxycycline. Tan and his colleagues [22] in Singapore found that resistance to tetracycline was 12% of isolates. The percentage of resistance for both trimethoprim-sulfamethoxazole and rifampin was 66%. Rifampin resistance inEscherichia coliandS. aureusis due to alterations in the target leading to a reduced affinity of the enzyme for the antibiotic [31].


## 3.6. Results of Antibiotic Resistance by MIC

In this study, 11 of 15 (73.3%)S. lugdunensisisolates were resistant to ampicillin (≥128 μg/mL), while 4 of 15 were having MIC values reached 2 μg/mL. The MIC values ofS. lugdunensisisolates against oxacillin revealed that 5 of 15 isolates reached 32 μg/mL, while MIC value of 2 isolates was ≥ 64 μg/mL. Six oxacillin resistant isolates (S1, S2, S3, S4, S5, and S6) hadmecAgene, but one isolate (S7) did not have such gene. Out of 15S. lugdunensisisolates (detected by MIC method), 14 isolates (93.2%) were sensitive to vancomycin, while only one isolate (6.8%) showed reduced susceptibility to vancomycin 8 μg/mL (intermediate resistant). Bourgeois and his coworkers [31] found that 6/13S. lugdunensisisolates were tolerant to vancomycin. No isolates showed any degree of resistance to vancomycin as in many data. ThevanA genes that are responsible for resistance (vangenes) are inducible and transferable and confer high-level resistance to vancomycin [32].


## 3.7.β-Lactamase Production

Eight isolates (53.3%) wereβ-lactamase producer. All these isolates were ampicillin resistant; seven of eight β-lactamase producing isolates were oxacillin resistant, while the remaining one was oxacillin sensitive. Six of eight were havingmecAgene (Table 2).Table 2
Relationship between ampicillin, oxacillin resistance with presence ofmecAgene, and β-lactamase production in Staphylococcus lugdunensisisolates.
Isolate number
Ampicillin resistant
Oxacillin resistant
mecA
β-Lactamase production
S1
+
+
+
+
S2
+
+
+
+
S3
+
+
+
+
S4
+
+
+
+
S5
+
+
+
+
S6
+
+
+
+
S7
+
+
−
+
S8
+
−
−
−
S9
+
−
−
−
S10
+
−
−
−
S11
+
−
−
+
S12
−
−
−
−
S13
−
−
−
−
S14
−
−
−
−
S15
−
−
−
−Mateo and her coworkers [24] found that 11.8% ofS. lugdunensiswere β-lactamase producers. Several authors reported that the percentages of β-lactamase-positiveS. lugdunensis vary from 24 to 40% in US isolates collections [33]. Papapetropoulos and his colleagues [34] isolated 14S. lugdunensisstrains from clinical specimens (abscesses and wounds) of 250 bed-hospital in Athens, Greece; 5 (30.2%) ofS. lugdunensiswere β-lactamase positive. The difference between this study and other studies may be due to the fact that the global using of β-lactam antibiotics in Iraq may result in induction of bacterial resistance to β-lactams via production of β-lactamases [35].


## 3.8. Detection ofmecAGene in MRSL Isolates

In this studymecAgene was detected in 6 (40%) of 15S. lugdunensis(Figure 5). All of these 6 isolates (S1, S2, S3, S4, S5, and S6) were resistant to oxacillin (Table 2). One isolate (S7) was resistant to oxacillin, butmecAwas not detected in this isolates.Gel electrophoresis of PCR ofmecA amplicon (499 bp) product: Lane L: ladder (1000-bp ladder); Lanes S1, 2, 3, 4, 5, and 6:mecA positive; Lanes S7, 8, 9, 10, 11, 12, 13, and 14:mecA negative samples.
(a)
(b)Staphylococcus lugdunensisis generally considered to be susceptible to oxacillin. Several studies reported negative PCR results when screening formecA, but among reports in the English literaturemecAhas been detected in twoS. lugdunensisisolates [8, 36]. Tee and his colleagues [8] reported a case of MRSL causing bloodstream infection in a neonate with an oxacillin MIC > 256 mg/L havingmecAgene. In 2008, Tan and his colleagues [22] found five (4.7%)S. lugdunensisstrains carrying themecAgene in a collection of 106 clinical isolates.


## 3.9. Antibiotic Susceptibility and Detection ofmecA Gene in“S. pseudolugdunensis” Isolates

The antibiotic susceptibility profile of“S. pseudolugdunensis” isolates (number = 7) is shown in Table 3. In addition to the results discussed earlier, this study represents the first record of characterization of oxacillin resistant andmecA positive“S. pseudolugdunensis” isolates recovered from clinical specimens in Iraqi hospitals (Table 4).Table 3
Antibiotic susceptibility profile of“S. pseudolugdunensis” isolates.
Isolate number
Ceftazidime-clavulanate
Ceftriaxone
Azithromycin
Imipenem
Doxycycline
amikacin
Cefixim
Amoxiclav
Vancomycin
Clindamycin
Rifampicin
TMP-SMZ
SP1
S
S
S
S
R
R
R
S
S
R
S
R
SP2
S
S
S
S
R
S
R
S
S
S
S
S
SP3
R
R
R
S
R
R
R
S
S
R
R
R
SP4
S
S
S
S
R
S
S
R
S
S
S
R
SP5
R
R
S
R
R
S
S
S
S
S
S
R
SP6
S
R
S
S
S
S
R
S
S
R
S
S
SP7
S
S
S
S
S
R
R
R
S
S
S
STable 4
Relationship between oxacillin resistance with presence ofmecA gene and β-lactamase production in “S. pseudolugdunensis” isolates.
Isolate number
Resistance to
β-Lactamase production
mecAgene
Ampicillin
Oxacillin 4μg/ml
Oxacillin  6μg/ml
Cefoxitin
SP1
+
+
+
−
+
−
SP2
+
+
+
+
+
+
SP3
+
+
+
+
+
+
SP4
+
+
+
+
+
+
SP5
+
+
−
−
−
−
SP6
+
+
+
+
+
+
SP7
+
−
−
−
−
−


## 4. Conclusion

The highest percentages ofS. lugdunensis isolates were recovered from subaxillary, wound swabs, and skin swabs samples, so this study reinforces the propensity ofS. lugdunensisto be associated with acute cutaneous infections. Although many other reports stated thatmecA gene presents low percentages inS. lugdunensis; however, the present study found high rate ofmecA in these bacteria.



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*Source: 736259-2014-07-13.xml*