Journal of Chemotherapy

ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20

Environmental Staphylococcus aureus contamination in a Tunisian hospital Haythem Gharsa, Raoudha Dziri, Naouel Klibi, Sarra Chairat, Carmen Lozano, Carmen Torres, Ridha Bellaaj & Karim Ben Slama To cite this article: Haythem Gharsa, Raoudha Dziri, Naouel Klibi, Sarra Chairat, Carmen Lozano, Carmen Torres, Ridha Bellaaj & Karim Ben Slama (2016) Environmental Staphylococcus aureus contamination in a Tunisian hospital, Journal of Chemotherapy, 28:6, 506-509, DOI: 10.1179/1973947815Y.0000000036 To link to this article: http://dx.doi.org/10.1179/1973947815Y.0000000036

Published online: 22 Jul 2016.

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Date: 12 December 2016, At: 18:27

Brief Communication

Environmental Staphylococcus aureus contamination in a Tunisian hospital Haythem Gharsa1, Raoudha Dziri1, Naouel Klibi1, Sarra Chairat1, Carmen Lozano2, Carmen Torres2, Ridha Bellaaj3, Karim Ben Slama1,4 1

Laboratoire des Microorganismes et Biomole´cules actives, Faculte´ de Sciences de Tunis, Universite´ de Tunis El Manar, Tunis, Tunisie, 2A´rea de Bioquı´mica y Biologı´a Molecular, Universidad de La Rioja, Logron˜o, Spain, 3 Military Hospital of Tunis, Montfleury, Tunisia, 4Institut Supe´rieur des Sciences Biologiques Applique´es de Tunis, Universite´ de Tunis El Manar, Tunis, Tunisie One hundred hospital environment samples were obtained in 2012 in a Tunisian hospital and tested for Staphylococcus aureus recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST), spa-typing, agr-typing and Sma I-pulsed-field gel electrophoresis (PFGE) were performed. Two methicillin-resistant S. aureus (MRSA) isolates typed as: ST247-t052-SCCmec I–agr I were recovered from the intensive care unit (ICU). Ten samples contained methicillin-susceptible S. aureus (MSSA) and these samples were collected in different services, highlighting the presence of the tst gene encoding the toxic shock syndrome toxin as well as the luk ED, hla, hlb, hld and hlgv virulence genes in some of the isolates. In conclusion, we have shown that the hospital environment could be a reservoir contributing to dissemination of virulent S. aureus and MRSA. Keywords: Staphylococcus aureus, Methicillin-resistance, TSST, Environmental hospital, Tunisia

Staphylococcus aureus is an ubiquitous commensal bacterium of the skin and mucous membranes of humans and many animals. It has been also isolated from different environments, being in hospital settings where this species has high ability to survive during several months.1,2 Nosocomial infections caused by S. aureus constitute a serious health concern worldwide. Although generally this microorganism is associated with skin and soft tissue infections, it can also cause severe diseases, such as pneumonia, meningitis, or septicaemia.3 Staphylococcus aureus is able to produce a wide range of extracellular toxins and virulence factors and has a high capacity of acquisition of antibiotic resistance mechanisms. There is a great concern with respect to methicillin-resistant S. aureus (MRSA) since it is resistant to most of beta-lactams and is one of the most relevant nosocomial pathogens nowadays.4 Surveillance of nosocomial infections is a core activity for the prevention, especially in reanimation and intensive care units (ICU).5 The objective of this study was to determine the prevalence of MRSA and S. aureus in Tunis Military Hospital environment. We aimed at the determination

Correspondence to: Karim Ben Slama, Laboratoire des Microorganismes et Biomole´cules Actives, Faculte´ de Sciences de Tunis, Universite´ de Tunis El Manar, 2092 Tunis, Tunisie. Email: [email protected]

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of resistance mechanisms, virulence traits and genetic lineages of the obtained isolates using different molecular techniques, including clonal diversity. A total of one-hundred environmental samples were taken in different services of the Tunis Military Hospital (Tunisia) during the period of March to April 2012, (services/samples): (Emergency/5; Haemodialysis Service/9; ICU/10; Obstetrics and Gynaecology/10; Paediatrics/6; Gastroenterology/16; Urology/9; Pneumology/7; Internal Medicine/7; Cardiology/5; Visceral Surgery/5; Neonatology/6; and Ophthalmology/5. Samples were cultured on Tryptone Soy Broth (TSB) and later streaked on Baird Parker agar (BPA) and Oxacillin-Resistance Screening Agar Base (ORSAB) supplemented with oxacillin (2 mg/L) and incubated at 37uuC for 24–48 hour, for S. aureus and MRSA recovery, respectively, One S. aureus suspected colony per positive plate was selected and identified by conventional methods [Gram-staining, catalase and oxidase tests, DNase production, and ability to coagulate rabbit plasma (BioRad)]. Staphylococcus aureus identification was then confirmed by amplification of the speciesspecific nuc gene.6 Staphylococcus aureus isolates were tested for susceptibility to 18 antimicrobial agents using the disc-diffusion method.7 Antimicrobial agents tested were as follow (charge in mg/disc): penicillin (10 U), oxacillin (1), cefoxitin (30),

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kanamycin (30), gentamicin (10), tobramycin (10), tetracycline (30), chloramphenicol (30), trimethoprim–sulphamethoxazole (1.25/23.75), erythromycin (15), clindamycin (2), amikacin (30), ciprofloxacin (5), mupirocin (5), vancomycin (30), teicoplanin (30), fusidic acid (10), and streptomycin (10 U). Double-disc diffusion test (D-test) with erythromycin and clindamycin discs was implemented in all isolates to detect inducible clindamycin resistance. The ribosomal methylases encoded by erm(A), erm(B), and erm(C) genes, which confer resistance to erythromycin and clindamycin, and the efflux pump encoded by msr(A) gene, conferring resistance to erythromycin, were studied by PCR in erythromycin-resistant isolates. In addition, tet(K), tet(M) and tet(L) genes conferring resistance to tetracycline, bla(Z) gene to penicillin, fus B and fusC genes to fusidic acid, and aph(3’)-IIIa gene to kanamycin, were studied by PCR in all antimicrobial resistant S. aureus isolates. Mutations in elongation factor G were studied by sequence analysis of fusA gene in all fusidic acid-resistant isolates.6 Recovered MRSA isolates were characterized by multi-locussequence-typing (MLST) (http://saureus.mlst.net/ ), and staphylococcal cassette chromosome mec (SCCmec) typing. The presence of the mecA gene was screened by PCR in all oxacillin and/or cefoxitin-resistant isolates.6 All S. aureus isolates recovered were characterized by determination of agr- and spa-type.8,9 Clonal relatedness of isolates was carried out by pulsed-field-gel-electrophoresis (PFGE) of chromosomal DNA previously digested with Sma I enzyme.6 The presence of leukocidin genes luk F/lukS-PV (encoding PantonValentine-leukocidin, PVL), luk ED and lukM, the haemolysin genes hla, hlb, hld, hlg, and hlgv, and exfoliating genes eta and etb, in addition to the tst gene was studied by PCR.6 Staphylococcus aureus isolates were recovered from 12 of the 100 environmental tested samples (12%) obtained from different services of Tunis Military Hospital. Methicillin-resistant S. aureus were detected in two of the 100 tested samples (2%) and MSSA in 10 of the samples (10%). The characteristics of the 12 S. aureus isolates recovered in this study are shown in Table 1 and Fig. 1. Contamination levels of hospital services by MSSA and MRSA showed less meaningful values than those already reported10 describing detection of S. aureus and MRSA in 63 and 46.7% of hospital environment surfaces, respectively. In another study performed in Japan,11 the rate of S. aureus and MRSA contamination was 27 and 8.7% in door handles, respectively. Previous works1,12,13 carried out in hospital environments showed a higher prevalence of MRSA, 74% in United Kingdom and 27–49% in United States of America.

2

Similarly to results of our study, the study of John et al. showed a low prevalence of MRSA, approximately 1%. Although, in our study the number of S. aureus isolated remained low, these isolates were detected in four different hospital units: two MRSA in ICU, five MSSA in Gastroenterology service, three MSSA in emergency service, and two MSSA in Obstetrics and Gynaecology service, showing a large distribution of S. aureus in different areas of the hospital. Similar results were obtained in a previous study.11 Our two MRSA strains were typed as ST247-t052-SCCmec I and agr type I. It is of interest to remark that the clone ST247SCCmec I-agr I detected in our MRSA isolates was also identified among healthcare-associated MRSA strains in Tunisian university hospitals, however, these strains presented different spa-type.14 The two MRSA isolates showed an identical PFGE-pattern (A). These isolates showed resistance, in addition to beta-lactams, to streptomycin, kanamycin, erythromycin, clindamycin, tetracycline, fusidic acid, tobramycin, gentamycin, amikacin and ciprofloxacin and harboured the genes bla(Z), erm(A), tet(M), aac(69)-aph(20), and aph(39)IIIa. No mutations in fusA gene that could justify this resistance phenotype were identified in fusidic-acidresistant MRSA isolates and they did not carry the fus B or fusC genes. Additionally, they also harboured the genes encoding alpha, beta, delta and gamma variant haemolysins, and the Luk-ED leukocidin. The three Hospital Services with detection of MSSA isolates were: Gastroenterology (5 of 16 samples), Emergency (3 of 5 samples) and Obstetrics and Gynaecology (2 of 10 samples). These isolates were recovered from different inanimate surfaces (door handles, sink, radiator, gastric endoscope and tap). Remarkably, the five isolates detected in the Gastroenterology unit showed the same PFGE pattern and also the same spa and agr type (t9606-agr I). The dissemination of this MSSA isolate in this area of the hospital has probably occurred, and special care should be taken to avoid an outbreak. The MSSA isolates recovered from different hospital services showed a high susceptibility to tested antibiotics (except penicillin, carrying bla(Z) gene). Interestingly, 60% of MSSA isolates harboured the tst gene encoding the toxic shock syndrome toxin. All MSSA isolates harboured the hla and hld genes, and lukED, hlgv, hlg and hlb genes were detected with a percentage ranging from 20 to 80% of isolates. In conclusion, we have shown that the hospital environment could be a reservoir contributing to dissemination of S. aureus and MRSA. More studies should be performed in the future to ameliorate our knowledge about the genetic lineages of S. aureus circulating in the hospital environment, as well as about the capacity of these strains to produce virulence

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Gastric 02/04/2012 endoscope Room door 05/03/2012 handles Tap 05/03/2012

Sink Care table

Sheet made

C5686

C5689

C5688 C5779

C5787

C5687

Tap

C5685

25/04/2012

05/03/2012 10/04/2012

02/04/2012

02/04/2012

Radiator

C5684

15/03/2012

11/03/2012

C5683

Toilet door handles

Wall

Room door 02/04/2012 handles Sink 02/04/2012

C5682

C5681

C5680

Strain

A

A

Emergency Obstetrics and Gynaecology Obstetrics and Gynaecology

Emergency

Emergency

D

B E

C2

C1

Gastroenterology F

Gastroenterology F

Gastroenterology F

Gastroenterology F

I

I

t377

t223 t197

t127

t127

I

I I

III

III

t9606 I

t9606 I

t9606 I

t9606 I

ST247 SCCmec I hla, hlb, hld, luk ED, hlgv ST247 SCCmec I hla, hlb, hld, luk ED, hlgv tst, hla, hld, hlg tst, hla, hld, hlg tst, hla, hld, hlg tst, hla, hld, hlg tst, hla, hld, hlg hla, hld, hlgv,luk ED hla, hld, hlgv,luk ED tst, hla, hld hla, hlb, hld, hlgv hla, hlb, hld, hlgv

Virulence agrSCCmec- gene type MLST type detected

t9606 I

t052

t052

PFGE- spatypeb type

Gastroenterology F

Intensive Care Unit

Intensive Care Unit (ICU)

Isolation date (day/month/year) Services

bla(Z)

P

bla(Z)

P

bla(Z) bla(Z)

bla(Z)

P, OXA

P, OXA P

bla(Z)

bla(Z)

bla(Z)

bla(Z)

bla(Z), tet(M), erm(A),aac(69)-aph(20), aph(39)-IIIa, fus(A) bla(Z), tet(M), erm(A), aac(69)-aph(20), aph(39)IIIa, fus(A) bla(Z)

Resistance genes detected

P

P, OXA

P

P

FOX, OXA, P, TB, GM, AK, K, TE, S, E, CC, CIP, FA FOX, OXA, P, GM, TB, AK, K, TE, S, E, CC, CIP, FA P, OXA

Phenotype of antibiotic resistancec

a: MRSA: methicillin-resistant S. aureus; MSSA: methicillin-susceptible S. aureus b: PFGE: pulsed-field gel electrophoresis c: FOX: cefoxitin; OXA: oxacillin; P: penicillin; E: erythromycin; TB: tobramycin; TE: tetracycline; K: kanamycin; AK: amikacin; S: streptomycin; CC: clindamycin; CIP: ciprofloxacin; FA: fusidic acid; GM: gentamicin

MSSA

MRSA

S. aureus a

Sample origin

Table 1 Characteristics of S. aureus strains detected in this study

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Figure 1 Dendrogram of pulsed-field gel electrophoresis Sma I patterns among 12 S. aureus isolates recovered from the hospital environment, generated by GelCompar II software using the UPGMA algorithm and the Dice similarity coefficients.

factors. Moreover, we must take into consideration the risk of hospital cross-infection and the acquisition and dissemination of the SCCmec element, which is responsible for methicillin resistance.

Acknowledgements This study was financed in part by project of the Tunisian Ministry of Higher Education and Scientific Research and also by project SAF2012-35474 of the Ministerio de Economia y Competitividad of Spain. Carmen Lozano has a contract associated with Project SAF2012-35474.

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Disclaimer Statements Contributors No contributors. Funding Tunisian Ministry of Higher Education and Scientific Research and project SAF2012-35474 of the Ministerio de Economia y Competitividad of Spain. Conflicts of interest There are no conflicts of interest.

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Ethics approval Ethical approval not required. 12

References 1 Boyce JM, Potter-Byno G, Chenevert C, King T. Environmental contamination due to methicillin-resistant Staphylococcus aureus: possible infection control implications. Infect Control Hosp Epidemiol. 1997;18:622–7. 2 Bergdoll MS, Lee Wong AC. Staphylococcal intoxications. Foodborne Infect Intox. 2006;3:523–52. 3 Aires de Sousa M, De Lencastre H. Evolution of sporadicisolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community acquired MRSA. J Clin Microbiol. 2003;41:3806–15. 4 Bouchami O, Achour W, Ben Hassen A. Typing of staphylococcal cassette chromosome mec encoding methicillin

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resistance in Staphylococcus aureus strains isolated at the bone marrow transplant centre of Tunisia. Curr Microbiol. 2009;59:380–5. Ben Jaballah N, Bouziri A, Kchaou W, Hamdi A, Mnif K, Belhadj S, et al. Epidemiology of nosocomial bacterial infections in a neonatal and pediatric Tunisian intensive care unit. Med Mal Infect. 2006;36:379–85. Gharsa H, Ben Sallem R, Ben Slama K, Go´mez-Sanz E, Lozano C, Jouini A, et al. High diversity of genetic lineages and virulence genes in nasal Staphylococcus aureus isolates from donkeys destined to food consumption in Tunisia with predominance of the ruminant associated CC133 lineage. BMC Vet Res. 2012;8:203. CLSI. Clinical Laboratory Standards Institute performance standards for antimicrobial susceptibility testing; eighteenth informational supplement. document M100-S22E. Wayne, PA: Clinical Laboratory Standards Institute; 2012. Shopsin B, Herring S, Kreiswirth BN. Hospital-acquired and community-derived: the future of MRSA? Clin Infect Dis. 2003;37:151–2. Harmsen D, Claus H, Witte W, Roth Ganger J, Claus H, Turnwald D, et al. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 2003;4:5442–8. Asoh N, Masaki H, Watanabe H, Watanabe K, Mitsusima H, Matsumoto K, et al. Molecular characterization of the transmission between the colonization of methicillin-resistant Staphylococcus aureus to human and environmental contamination in geriatric long-term care wards. Intern Med. 2005;44:41–5. Oie S, Hosokawa I, Kamiya A. Contamination of room door handles by methicillin-sensitive/methicillin-resistant Staphylococcus aureus. J Hosp Infect. 2002;51:140–3. Bures S, Fishbain JT, Uyehara CFT, Parker JM, Berg BW. Computer keyboards and faucethandles as reservoirs of nosocomial pathogens in the intensive care unit. Am J Infect Control. 2000;28:465–71. French GL, Otterb JA, Shannon KP, Adams NM, Watling D, Parks MJ. Tackling contamination of the hospital environment by methicillin-resistant Staphylococcus aureus (MRSA): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination. J Hosp Infect. 2004;57:31–7. Ben Joma`a-Jemili M, Teruyo I, Meng Z, Zhang M, Jin J, Li S, et al. Molecular characterization of methicillin-resistant Panton-valentine leukocidin positive Staphylococcus aureus clones disseminating in Tunisian hospitals and in the community. BMC Microbiol. 2013;13:2.

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Environmental Staphylococcus aureus contamination in a Tunisian hospital.

One hundred hospital environment samples were obtained in 2012 in a Tunisian hospital and tested for Staphylococcus aureus recovery. Antimicrobial res...
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