Variable number of tandem repeat profiles and antimicrobial resistance patterns of Staphylococcus haemolyticus strains isolated from blood cultures in children

    Variable number of tandem repeats profiles and antimicrobial resistance patterns of Staphylococcus haemolyticus strains isolated from blood cultures in children Faride Hosseinkhani, Fereshteh Jabalameli, Narges Nodeh Farahani, Morovat Taherikalani, Willem B. van Leeuwen, Mohammad Emaneini PII: DOI: Reference:

S1567-1348(15)30065-4 doi: 10.1016/j.meegid.2015.11.035 MEEGID 2568

To appear in: Received date: Revised date: Accepted date:

2 August 2015 2 November 2015 30 November 2015

Please cite this article as: Hosseinkhani, Faride, Jabalameli, Fereshteh, Farahani, Narges Nodeh, Taherikalani, Morovat, van Leeuwen, Willem B., Emaneini, Mohammad, Variable number of tandem repeats profiles and antimicrobial resistance patterns of Staphylococcus haemolyticus strains isolated from blood cultures in children, (2015), doi: 10.1016/j.meegid.2015.11.035

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ACCEPTED MANUSCRIPT Variable number of tandem repeats profiles and antimicrobial resistance patterns of Staphylococcus haemolyticus strains isolated from blood cultures in children Hosseinkhani1,

Fereshteh

Jabalameli1,

Narges

Nodeh

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1

Morovat

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Taherikalani3, Willem B. van Leeuwen4, Mohammad Emaneini1*

Farahani2,

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Faride

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences,

Tehran, Iran.

Children's Medical Center, Tehran University of Medical Science, Tehran, Iran

3

Department of Microbiology, School of Medicine, Lorestan University of Medical Sciences,

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2

Khorramabad, Iran.

Leiden, the Netherlands. *

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Corresponding author:

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Department of Innovative Molecular Diagnostics, University of Applied Sciences Leiden,

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E-mail: [email protected]

Tel- Fax: 098- 021- 8895- 5810 Address:

Department of Microbiology, School of MedicineTehran University of Medical Sciences 100 Poursina St., Keshavarz Blvd., Tehran, Iran

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ACCEPTED MANUSCRIPT ABSTRACT Staphylococcus haemolyticus is a healthcare-associated pathogen and can cause a variety of

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lifethreatening infections. Additionally, multi-drug resistance (MDR), in particular

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methicillin-resistant S. haemolyticus (MRSH) isolates, have emerged. Dissemination of such

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strains can be of great concern in the hospital environment. A total number of 20 S. haemolyticus isolates from blood cultures obtained from children were included in this study. A high prevalence of MDR-MRSH isolates with high MIC values to vancomycin was found

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and 35% of the isolates were intermediate resistant to vancomycin. Multilocus variable

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number of tandem repeats analysis (MLVF) revealed 5 MLVF types among 20 isolates of S. haemolyticus. Twelve isolates shared the same MLVF type and were isolated from different

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wards in a pediatric hospital in Iran. This is a serious alarm for infection control; i.e. in the

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absence of adequate infection diagnostics and infection control guidelines, these resistant

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strains can spread to other sectors of a hospital and possibly among the community.

patients.

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Keywords: S. haemolyticus, MLVF typing, multidrug resistance, blood culture, pediatric

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ACCEPTED MANUSCRIPT 1. Introduction

Staphylococcus haemolyticus (SH) isolates have the ability to colonize the human skin and

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mucosal membranes (Cavanagh et al., 2012) and is the primary cause of catheter-related bloodstream infections (Flahaut et al., 2008; Takeuchi et al., 2005 Cavanagh et al., 2012).

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The ability of SH to develop multidrug-resistance (MDR), including to glycopeptides, represents a serious threat (Blavasco et al., 2000; Cavanagh et al., 2012; Flahaut et al., 2008).

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The genome of SH contains large amounts of mobile genetic elements coding for antimicrobial resistance, which can be transferred to related staphylococcal species (Vollú

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Silva et al., 2013). Colonized healthcare workers and medical devices may serve as a reservoir for transmission (Vollú Silva et al., 2013). Epidemiological analysis and

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identification of the antibacterial resistance pattern of this staphylococcal species are

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mandatory for appropriate infection control measurements (Hope et al., 2008).

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Multilocus variable number of tandem repeats analysis (MLVF) is used to identify possible genetic associations among strains. MLVF is based on natural variation in the number of tandem DNA repeats, as present in multiple loci of most bacterial genomes and used as a

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phylogenetic marker (Cavanagh et al., 2012). This method is simple, rapid and highly discriminatory (Holmes et al., 2010). In many microbiological diagnostic laboratories worldwide, SH is not identified at the species level. Hence, no informative data is available to address basic questions concerning its population structure (Becker et al., 2014). The aim of this study was to evaluate the antimicrobial resistance of SH isolated from children's blood cultures and to identify possible transmission of isolates among patients in a pediatric hospital.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1. Strain collection

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In total 20 SH strains isolated from blood cultures obtained from 18 children (10 children

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between 2-5 years, average 3.4 years and 8 newborns between 10 days throught 11 months, average 99 days) hospitalized at Children's Medical Center Tehran, Iran during the period 2013 to 2014 were included. SH strains were obtained from patients hospitalized for more

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than 3 days (85%) and outpatients (15%).

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Isolates were identified to the species level using standardized biochemical methods (Winn et al., 2006) and subsequently confirmed using a PCR-method targeting thermonuclease (nuc)

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gene as described previously (Hirotaki et al., 2011).

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2.2. Antibiotic susceptibility testing

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All isolates were tested for cefoxitin, ciprofloxacin, clindamycin, erythromycin, gatifloxacin, gentamicin, linezolid, rifampin, synercid (quinopristin-dalfopristin), tetracycline and

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trimethoprim/sulfamethoxazole using the disc agar diffusion method according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2013, M100-S23). Minimum Inhibitory Concentration of all bacterial isolates to vancomycin and oxacillin was determined by the micro broth dilution method as recommended by CLSI (2013, M100-S23). S.aureus ATCC 29213 and Enterococcus faecalis ATCC29212 were used as reference strains. Multidrugresistance was defined as resistance of the bacterial strain to three or more classes of antimicrobials (Cavanagh et al., 2012). 2.3. DNA extraction and mecA PCR Template DNA was generated by boiling method as described previously (Fatholahzadeh et al., 2008). DNA was stored at −20 °C until use. Presence of the mecA gene was analaysed for 4

ACCEPTED MANUSCRIPT all phenotypically oxacillin-resistant isolates using a PCR protocol as described previously (Blavasco et al., 2000).

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2.4. MLVF typing

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MLVF types were defined based on the difference in the 5 loci (L1-L5) and was performed as previously described. Isolates with a VNTR pattern difference in no more than one locus are considered the same type (Cavanagh et al., 2012).

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3. Results

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Antibiotic resistance patterns and molecular characterization of isolates are presented in Table 1. All isolates were susceptible to vancomycin, however 35% of the isolates

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demonstrated intermediate resistance (Table 2). The MIC50 and MIC90 of the isolates for

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vancomycin were 4mg/l and 8mg/l respectively. All strains were phenotypically oxacilin

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resistant (MIC50 and MIC90 values of 32mg/l and 128mg/l respectively). Only 80% of the isolates harbored the mecA gene and were defined as MRSH isolates. Overall, 85% of the

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isolates were MDR.

Among the 5 loci studied in MLVF typing, no DNA fragment was observed in locus L5. Variable number of tandem repeats (VNTR) pattern analysis revealed 13 unique patterns and 5 different MLVF types among all SH isolates. The most predominant type (type I) included 12 isolates. 5. Discussion Worldwide emergence of SH as a primary cause of infection has been reported (Becker et al., 2014; Gupta et al., 2012). Moreover, multidrug resistance has posed significant burden in the treatment of SH infections (Gupta et al., 2012). In this study, the frequency of MDR-SH isolates was high (85%), which is confirmed by reports from different parts of the world 5

ACCEPTED MANUSCRIPT (Becker et al., 2014). No mecA gene was detected in 20% of the phenotypically oxacillinresistant SH isolates. Possible explanation may be the presence of a novel mecA allotype,

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beta-lactamase hyperproduction or alteration in genes encoding other penicillin binding

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proteins (Ba et al., 2014, Barros et al., 2012). Most of SH isolates were susceptible to

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rifampin and quinopristin-dalfopristin as reported previously (Becker et al., 2014). However, these antimicrobial agents are not recommended for treatment of blood infections in children or neonates according to their toxicity (Liu et al., 2011). In our study, resistance to

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tetracycline was significantly higher than mentioned in Europan and US studies (Coenen et

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al., 1997). This could be a possible effect of a more strict antibiotic policy in these countries (Becker et al., 2014). Although all isolates were susceptible to vancomycin, it is noteworthy

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to mention that the MIC level was high (MIC50: 4mg/l, MIC90: 8mg/l) and confirmed by

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previous studies from different part of the world (Barros et al., 2012; Becker et al., 2014; Vollú Silva et al., 2013). Latter phenomenon is a serious warning for infection control in the

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hospital environment. The extensive use of this antibiotic may cause the emergence of vancomycin-resistant strains (Emaneini et al., 2007; Kim et al., 2012; Kresken et al., 2009).

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The high discriminatory power qualifies MLVF as a convenient typing method for the classification of SH isolates, which has a rather clonal population structure. High frequency of MLVF type I isolates and distribution of these isolates in the different wards of the hospital, indicating the presence of a common MDR clone in the hospital environment (Luczak-Kadlubowska et al., 2008). In conclusion, this study revealed the high frequency of MDR-MRSH among different wards of an Iranian children’s hospital. In high-risk patients (neonates and immuno-compromised patients) infection with resistant clones can significantly increase the morbidity and mortality and a prolonged stay in the hospitals. (Cavanagh et al., 2012; Srinivasan et al., 2002; Forbes et al., 2007). This is a serious alarm for infection control; i.e. in the absence of proper and 6

ACCEPTED MANUSCRIPT suitable microbiological diagnostics and infection control guidelines, these resistant strains can spread to other sectors of a hospital and possibly among the community. Moreover, this

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species can act as a reservoir for the transmission of antibiotic resistance markers to other

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related and clinically relevant species, such as S. aureus (Becker et al., 2014).

Conflict of interests

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The authors declare that there is no conflict of interest to reveal. Acknowledgments

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This project has been financially supported by Tehran University of Medical Sciences

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and Health Services, Tehran, Iran. Study grant no: 25170/93-02-30.

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Staphylococcus

haemolyticus

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Genome and the Evolution of Human-Colonizing Staphylococcal Species. J Bacteriol. 187, 21, 7292-308.

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Vollú Silva, P., Cruz R. S., Keim, L. S., Renato de Paula, G., Carvalho, B. T. F., Coelho, L.

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ACCEPTED MANUSCRIPT Table1. Antimicrobial resistance patterns and variable number of tandem repeats profile of Staphylococcus haemolyticus strains isolated from children's blood cultures VNTR1 loci (bp) Isolate*

MLVF

Ward** L1

L2

L3

L4

2

Resistance pattern

mecA

MDR†

+

+

+

+

+

+

ERY, CLN, CIP, RIP, GAT, TS, GM, OXA

+

+

ERY, ,CIP, GAT, TS, GM, OXA

+

+

T, OXA, ERY, CLN

-

+

TS, OXA

+

‫ــ‬

type

EMR

500

500

700

100

ERY#, CLN, CIP, RIP, GAT, TS, GM, OXA

2

NICU

500

500

700

100

ERY, CLN, T, TS, GM, OXA

3

EMR

500

500

700

100

ERY, CIP, RIP, GAT, TS, GM, OXA

4

NICU

500

500

700

100

5

NEURO

500

500

-

100

6

INF

500

500

-

100

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T

1

500

500

400

100

8

NEPH

500

500

500

100

T, OXA, ERY

+

+

9

OP

500

500

500, 700

100

MG, OXA

+

‫ــ‬

10

EMR

500

-

700

100

ERY, CLN, SYN, CIP, GAT, TS, GM, OXA

+

+

11

EMR

500

300, 500

700

100

ERY, T, TS, OXA

+

+

12

EMR

500

300, 500

700

100

ERY, CLN, CIP, GAT, T, OXA

-

+

13

SUR

500

300, 500

200

100

ERY, CLN, CIP, T, TS, OXA

+

+

14

OP

500

300, 500

200

100

ERY, CLN, OXA

-

‫ــ‬

15

SUR

500

300, 500

200, 600

100

ERY, CLN, , SYN, T, TS, OXA

-

+

16

NEURO

500

300, 500

400

100

ERY, RIP, GAT, TS, GM, OXA

+

+

17

OP

500

300, 500

700

-

III

T, TS, OXA

+

+

18

NICU

500

300

200, 700

100

IV

ERY, CLN, CIP, GAT, TS, GM, OXA

+

+

19

INF

500

-

600

100

ERY, CIP, GAT, TS, GM, OXA

+

+

20

NEPH

500

-

600

100

ERY, CIP, GAT, TS, OXA

+

+

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EMR

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* Strains 5 and 16 were recovered from one patient and strains 13 and 15 from the another patient. ** EMR: emergency; NICU: neonatal intensive care unit; NEURO: neurology; INF: infant; NEPH: nephrology; OP: outpatient; SUR: surgery 1

VNTR: Variable Number of Tandem Repeat.

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MLVF: Multi Locus Variable number of tandem repeats Fingerprinting

#

ERY, erythromycin; CLN, clindamycin; SYN, synercid;،CIP, ciprofloxacin; RIP, rifampin; GAT, gatifloxaci; T, Tetracycline; TS; trimethoprim-sulfamethoxazole; GM,

gentamicin; OXA, oxacillin. †

MDR: Multidrug resistant

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ACCEPTED MANUSCRIPT

Table 2. Distribution of vancomycin and oxacillin MIC values among

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Staphylococcus haemolyticus strains isolated from children’s blood cultures.

vancomycin oxacillin

Break point

≤0.5

1

2

R* ≥32 µg/ml

4***

-

3

R ≥0.5µg/ml

1

2

3

4

8

16

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Antibiotic

IP

MIC** Values (mg/l)

32

64

≥128

6

6

1

-

-

-

-

1

1

3

4

5

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* Resistance, breakpoint is selected according to CLSI guidelines; ** MIC50: Bold ; MIC90: Underlined

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*** The number corresponds to the number of isolates with a certain MIC-value

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ACCEPTED MANUSCRIPT

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High prevalence of MDR-Staphylococcus haemolyticus was detected in blood cultures of children.

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

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Highlights

35% of the isolates were intermediate resistant to vancomycin.

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All strains were susceptible to linezolid.

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5 different types among 20 isolates of S. haemolyticus were documented.

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Twelve isolates had same MLVF types and were isolated from different wards in a pediatric hospital of Iran.

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

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