Identification of Hemolysin encoding Genes and their Association with Antimicrobial Resistance Pattern among Clinical Isolates of Coagulase-Negative Staphylococci

doi:10.21203/rs.2.16458/v1

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Identification of Hemolysin encoding Genes and their Association with Antimicrobial Resistance Pattern among Clinical Isolates of Coagulase-Negative Staphylococci

https://doi.org/10.21203/rs.2.16458/v1

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published 10 Feb, 2020

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Objection : Coagulase-negative staphylococci (CoNS) are considered opportunistic pathogens which capable of producing several toxins, enzymes and also resistance genes. The current study aimed to determine the occurrence of different hemolysins and patterns of antibiotic resistance among CoNS species.

Results : The highest frequency of antibiotic resistance was observed against cefoxitin in 49 isolates (53.8%), and the lowest resistance was against novobiocin in 5 isolates (5.5%). None of the isolates were resistant to vancomycin. The prevalence of hla, hla_yidD, hld, and hlb genes were determined as 87.9%, 62.6%, 56%, and 47.3%, respectively. The hla/yidD and hld genes were detected in 69.4% of S. epidermidis and the hla gene in 94.6% of S. haemolyticus ; hlb gene was detected in 53.1% of the S. epidermidis isolates. mecA gene was identified in 50 (55%) of the CoNS isolates. In conclusion, the results of statistical analysis showed that the hld gene had a significant association with resistance to levofloxacin and erythromycin and the hlb with clindamycin resistance. The results of this study showed that there is a significant relationship between hemolysin encoding genes and antibiotic resistance patterns; therefore, detection of virulence factors associated with antibiotic resistance has become a significant issue of concern.

General Microbiology

CoNS

hemolysin

antibiotic resistance

Coagulase-negative staphylococci (CoNS) are considered opportunistic pathogens that cause a variety of infections, particularly among immunocompromised, long-term hospitalized patients, preterm infants and in patients with indwelling or different implant polymer bodies [1–3]. Among various CoNS species, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus have been confirmed to be responsible agents for the vast majority of nosocomial infections [4]. Treatment of CoNS infections has become more complicated, as many isolates in hospitals show high rates of resistance to multiple antimicrobial agents of clinical relevance [5]. CoNS is also a reservoir for resistance genes that can be transmitted to other pathogens [6]. About 80%–90% of CoNS isolates associated with nosocomial infections are methicillin-resistant coagulase-negative Staphylococci [7]. Most antibiotic resistance genes are carried on a plasmid and more often found in methicillin-resistant [8]. CoNS are capable of producing several toxins and enzymes characteristically associated with Staphylococcus aureus such as hemolysins, which are responsible for the invasion of host cells [9, 10].Hemolysins of staphylococci are categorized into four different types, including alpha (α), beta (β), gamma (γ), and delta (δ). The α-toxin is encoded by the hla, acts as pore-forming cytotoxin (PFT), which active against a wide array of human cells. Pathogenicity of this toxin is due to hemolytic, dermonecrotic, and neurotoxic effects [11–14]. β-toxin which encoded by the hlb gene is known as Mg2+-dependent sphingomyelinase. Incubation at temperatures below 10°C intensify the cytolytic activity of β-toxin; thus, it is often referred to as the ‘hot-cold’ hemolysin [15, 16]. The hld gene encodes a 26 amino acid peptide, which is referred to as delta (δ) hemolysin. This toxin with its detergent function degrades erythrocytes. The delta toxin may cause intestinal diseases that can vary from acute diarrhea to severe enteritis [15, 17, 18]. The current study aimed to determine the prevalence of various CoNS species, identify different types of hemolysins, analysis the probable association between the presence of hemolysins and antibiotic resistance.

1–1-Identification of CoNS isolates

A total of 91 CoNS isolates were collected from various clinical specimens submitted to three teaching hospitals (including Beheshti, Besat, and Farshchian) located in Hamedan, Iran, from September 2017 to January 2019. All isolates were investigated by conventional methods. The origins of the isolates were as follows: blood, urine, catheters, and wounds. This study was approved by the Ethics Committee of Hamadan University of Medical Sciences (Code No: IR.UMSHA.REC.1396.827).

1–2-Antibiotic susceptibility testing

Antibiotic susceptibility testing of 91 CoNS species carried out using the standard disk agar diffusion (DAD) method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [19] for the following antimicrobial agents: chloramphenicol (30 µg), cefoxitin (30 µg), clindamycin (2 µg), doxycycline (30 µg), erythromycin (15 µg), gentamicin (10 µg), levofloxacin (5 µg), novobiocin (5 µg), rifampicin (5 µg), trimethoprim-sulfamethoxazole (25 µg) and vancomycin (30 µg) (MAST, Merseyside, UK). S. aureus ATCC33591 was used as a quality control.

1–3-DNA extraction from isolates

CoNS Chromosomal DNA was extracted by boiling method. Quality of extracted DNA was assessed by the Nanodrop ND1000 (Nanodrop Technologies, Inc., Wilmington, DE, USA).

1–4-Detection of hemolysin genes

Identify of hemolysin encoding genes was performed by Multiplex PCR (hla haem, hla/yidD epid and hlb_epid) and single PCR (hld_epi) using specific primers (Table S1), The PCR conditions included an initial denaturation at 94 ºC for 4 min, followed by amplification; 30 cycles at 94 ºC for 1 min, 58 ºC for 1 min (45 ºC–1 min for hld_epi), 72 ºC for 1 min and 72 ºC for 5 min. hla_haem: S. haemolyticus ATCC 29970, hlb_epid: S. epidermidis ATCC 12228 and hla/yidD_epid: S. epidermidis ATCC 12228, were used as control.

1–5-Detection of mecA gene

PCR assay was used for the identification of mecA gene, using specific primers which presented in Table 1. Amplification involved an initial denaturation at 95°C for 10 minutes, followed by 35 cycles of denaturation (95°C for 30 seconds), annealing (55°C for 30 seconds), and extension (72°C for 60 seconds), with a final extension step (72°C for 7 minutes). S. aureus ATCC29247 was used as control.

1–6-Statistical analysis

The statistical method was used at determining the frequency, the relationship between the occurrence of hemolysins and antimicrobial resistance and also samples type. The data were analyzed by Chi-Square test, and the difference was considered statistically significant in P<0.05 by SPSS software version 20.

2–1-Isolation and prevalence of CoNS isolates

Of the 91 clinical isolates of CoNS, 49 (53.8), 37 (40.7%) and 5 (5.5%) isolates were recognized as S. epidermidis, S. haemolyticus, and S. saprophyticus, respectively. Among these isolates, 39 isolates (42.9%) from the blood, 33 isolates (36.3%) from the urine, 13 isolates (14.3%) of the catheter and six isolates (6.6%) were recovered from wound samples.

2–2-Antimicrobial susceptibility testing

The results of the antibiotic susceptibility testing of 91 CoNS isolates, including 50 isolates (55%) of Methicillin-resistant CoNS (MR-CoNS) and 41 isolates (45%) of Methicillin-susceptible CoNS (MS-CoNS) are presented in Table 1. Multi-drug resistance was identified among 96% (48 isolates) of MR-CoNS isolates and 63.4% (26 isolates) of MS-CoNS isolates. Among MR-CoNS strains, the most frequent resistant, was to cefoxitin (98%). Among CoNS species, the highest resistance was observed for cefoxitin (53.8 %,). None of the CoNS species was identified as being resistant to vancomycin.

2–3-Prevalence of hemolysin genes among CoNS isolates

The distribution of hemolysin genes among CoNS isolates were presented in Table 2. Our results showed a high frequency of hemolytic activity among CoNS strains isolated from blood, followed by urine, catheter, and wound, respectively. 80 isolates (87.9%) with hla gene, 57 isolates (62.6%) with hla/yidD_epid, 51 isolates (56%) with hld and 43 isolates (47.3%) with hlb. Only 15 isolates (16%) had one type of the hemolysin, included; 12, 1 and two hla, hlb and hla/yidD positive CoNS isolates, respectively. Two types of hemolysins were identified in 32 (35%) of the strains, hla+hla/yidD was the most abundant with frequency 13(14%), predominating followed by; hla+hld = 12(13%), hla+hlb = 4(0.04%), hld+ hla/yidD = 2 (0.02%) and hlb+ hla/yidD = 1(0.01%). 14(15%) of the strains were carrying three types of the hemolysins; hla+hlb+hla/yidD,hlb+hld+hla/yidD = 4(0.04%) and hla+hld+hla/yidD = 3 (0.03%). All hemolysin genes were detected in 23 (25%) of the identified isolates.

2–4-Prevalence ofmecAgene among CoNS isolates

The distribution of mec A gene among CoNS isolates and the prevalence of hemolysin genes among MR-CoNS species are presented in Table S2. S. epidermidis strains had the highest frequency of mecA gene. Among MR-CoNS isolates, hla gene was the most common at a rate of 90%, followed by hla_yiD(60%), hld(58%) and hlb(48%).

2–5-Statistical analysis

Statistical analysis results are presented in Table 3. Statistically, a significant association between CoNS species and the occurrence of hld and hla_yiD genes was observed in this study. It was found a meaningful relationship between antibiotic resistance and the presence of the genes for delta and beta hemolysins. In contrast to the beta and delta hemolysins, there was no significant correlation between the presence of genes for alpha-hemolysin and antibiotics.

In the present study, S. epidermidis (53.8%) was the most clinically significant of the CoNS species and S. haemolyticus (40.7%) was identified as the second, which is in agreement with other reports [20–22]. The rate of CoNS isolation was highest in blood samples (43%) followed by urine (36.3%), catheters (14.3%) and wounds samples (6.6%). Changdeo S.Aher et al., and Shadhvi Parashar et al., were also reported that the vast majority of CoNS species isolated from the blood than urine [23, 24]. In our study, the highest resistance rate of the CoNS was against methicillin (55%), followed by cefoxitin (53.8%) and the other ones. According to previous studies, which founded significant discrepancies in resistance ratio against cefoxitin from 58% to 84.7% and high resistance to erythromycin (76.9%) and Trimethoprim-sulfamethoxazole (74.9%) [7, 25–27]. In the current study, the resistance ratio to methicillin was determined in 50 (55%) of CoNS isolates; the findings were comparable to other studies [28–31], who reported resistance to methicillin with rates of 43.8%, 59.64%, 62.8%, and 73.3%, respectively. In the present study, the prevalence of the multiple antibiotic resistance was significantly higher among MR-CoNS isolates compared to methicillin-susceptible CoNS (MS-CoNS) isolates which is similar to the results which reported by F. Koksal et al., [31]. The results of our studies indicated that the mecA gene was identified in all of the CoNS strains revealing phenotypical resistance to cefoxitin, and only one strain of mecA-positive S. epidermidis was phenotypically cefoxitin-susceptible, which is in agreement with the others [27, 32]. Like other studies [25, 33], this study revealed that all CoNS isolates were also fully sensitive to vancomycin. However, there are reports of the occurrence of decreased susceptibility to vancomycin in CoNS isolates [34, 35]. Out of 91 CoNS strains, the percentage of strains with hemolytic activity were the highest among S. epidermidis (53.8%), followed by S. haemolyticus (39.5 %) which is in agreement with the findings of Cunha Mde L and Pinheiro L [36, 37], but, in contrary with the findings of Azih and Enabulele [20]. Among CoNS species, 94.6% and 81.6% of the S. haemolyticus and S. epidermidis strains were determined to be positive to the hla gene, respectively, and the hlb gene was detected in 53 % and 46% of the S. epidermidis and S. haemolyticus strains, respectively. On the other hand, according to Okee MS et al., the occurrence of hlb gene not observed in any of isolated CoNS species and hla gene found in only 20% of S. epidermidis, which is also in contrary to a similar survey carried out by Pinheiro L et al., who demonstrated the emergence of the hla and hlb genes in 92.9% of the S. epidermidis strains and hla at the same frequency (91.7%) in S. hemolyticus strains [37, 38]. In the present study, the largest number of the strains carrying the hld gene belonged to S. epidermidis with frequency of 69.4%, then S. hemolyticus (37.8%), according to Pinheiro et al., 95.3% of the S. epidermidis were carrying hld gene, and none of the S. haemolyticus [37]. In a comparative analysis performed among the MR-CoNS and MS-CoNS isolates, statistically founded no significant association between the emergence of the hemolysins and methicillin resistance (p-value from 0.472 to 0.962). All of the isolated MR-CoNS demonstrated hemolytic activity either alone or in combined forms, while only one of the MS-CoNS strains was nonhemolytic which belonged to a S. haemolyticus recovered from the urine sample. Among MR-CoNS, the highest number of strains carrying hemolysin genes are isolated from the blood rather than urine. But in a similar study done by Motamedi et al., S. aureus hemolytic isolates are identified more in blood and ulcers than urine and catheter samples [39]. Regarding the pattern of antimicrobial resistance in CoNS isolates found a significant association between the occurrence of hld gene and resistance to levofloxacin and erythromycin (p-value = 0.04) as well as between hlb and clindamycin resistance (p-value = 0.009). Our results showed that the frequency of hemolytic agent genes of hla, hlb, hld were higher in cefoxitin resistant isolates than susceptible isolates. The analysis of our results showed that there is no significant association between the type of clinical sample and hemolysins which is similar to the results of studies conducted by Corredor Arias et al., in S. aureus strains [40]. Arabestani et al., Lee et al., and Osman et al. proved significant associations between pathogenicity factors and antibiotic resistance [41–43].

The results of this study indicated an increasing resistance to methicillin and the other drugs in CoNS strains which demonstrated the critical role of antibiotic resistance. This study demonstrated that the hla and hla_yidD genes and resistance to methicillin, cefoxitin, and trimethoprim-sulfamethoxazole antibiotics are widely distributed among most of the CoNS isolated from a human.

The number of bacteria isolates was rather low, if the number of bacteria was more, the results would be better and also we have to design another study which focus on gene expression of the resistance and virulence factors.

Coagulase-negative staphylococci (CoNS), pore-forming cytotoxin (PFT), Brain Heart Infusion (BHI), disk agar diffusion (DAD), Clinical and Laboratory Standards Institute (CLSI), methicillin-susceptible CoNS (MS-CoNS)

Ethical Approval and Consent to participate

This study was approved by the ethics committee of the Hamadan University of Medical Sciences ((Code No: IR.UMSHA.REC.1396.827)) and about the consent to participate is not applicable.

Consent for publication

Its not applicable

Availability of supporting data

The supporting data files included table S1 and S2.

Competing interests

The authors declare that they have no competing interests

Funding

The Research and technology of Vice Chancellor of Hamadan University of Medical Sciences (Project number: 9612087936).

Authors’ contributions

MN and ZS performed the tests, collected and analyzed the data, BA was advisor and contributor in writing and editing the manuscript, GR, performed the analysis of the data.M.R A designed the project and contributes in the whole steps of the projects. All authors read and approved the final manuscript.

Acknowledgments

The authors of this article are grateful to Hamadan University of Medical Sciences for their financial support in conducting research. This work was supported by the vice chancellor and Research technology of Hamadan University of Medical Sciences on the grant number, 9612087936.

Authors’ Information:

Mona Nasaj, PhD candidate in Medical Microbiology, Microbiology department, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran, Zahra Saidi, MSc. Student in Medical Microbiology, Hamadan University of Medical Sciences, Hamadan, Iran. Babak Asgari, , Assistance Professor in Medical Bacteriology, Microbiology department, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran,. Ghodratollah Roshanaei, Associate Professor Department of Biostatistics, School of Health, Hamadan University of Medical Sciences, Hamadan, Iran. Mohammad Reza Arabestani^,Associate Professor in Medical Bacteriology, Microbiology department, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

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Table 1: Prevalence of antibiotic-resistant strains among MR-CoNS and MS-CoNS.

MS-CoNS (n=41)			MR-CoNS (n=50)*
S. saprophyticus (n=3)	S. haemolyticus (n=15)	S. epidermidis (n=23)	S. saprophyticus (n=2)	S. haemolyticus (n=22)	S. epidermidis (n=26)	Antimicrobial agent
0	0	0	2	22	25	Cefoxitin
1	5	10	1	10	15	Trimethoprim-sulfamethoxazole
1	4	9	0	8	4	Erythromycin
0	3	6	0	6	4	Clindamycin
0	2	6	0	7	8	Chloramphenicol
0	1	1	1	0	0	Riphampin
2	1	4	0	3	1	Levofloxacin
0	0	0	0	0	0	Vancomycin
0	2	2	0	6	10	Gentamicin
1	5	7	0	2	7	Doxycycline
3	0	0	2	0	0	Novobiocin

*MR, methicillin-resistant; CoNS, coagulase-negative staphylococci; MS, methicillin-sensitive.

Table 2: Prevalence of hemolysin encoding genes among various CoNS species and various clinical samples

	CoNS(n=91)				Source
Total n(%)	S. saprophyticus (n=5)	S. haemolyticus n=37))	S. epidermidis n=49))	Types of hemolysins	Blood n(%)	Urine n(%)	Wound n(%)	Catheter n(%)
80(87.9)	5(100)	35(94.6)	40(81.6)	hla	37(95)	29(88)	4(66.7)	10(77)
57(62.6)	0	23(62.2)	34(69.4)	hla/yiD	25(64.1)	18(54.5)	4(66.7)	10(77)
43(57.3)	0	17(46)	26(53.1)	hlb	18(46.2)	15(45.5)	4(66.7)	6(46.2)
51(56)	3(60)	14(37.8)	34(69.4)	hld	22(56.4)	18(54.5)	4(66.7)	7(53.8)

Table 3. Statistical analysis results for determining possible relationship between the following variables and the presence of types of hemolysins genes

Types of hemolysins

mecA gene

Clinical sample

CoNS species

different classes of antibiotics

Cli

Ery

Levo

Novo

hla

P=0.472

P=0.122

P=0.131

P=0.448

P=0.708

P=0.820

P=0.391

hla_yiD

P=0.627

P=0.545

P=0.009

P=0.178

P=0.659

P=0.398

P=0.003

hlb

P=0.962

P=0.807

P=0.075

P=0.009

P=0.183

P=0.881

P=0.028

hld

P=0.678

P=0.954

P=0.014

P=0.123

P=0.043

P=0.04

P=0.855

CoNS; coagulase-negative staphylococci, Cli; clindamycin, Ery; erythromycin, Riph; rifampicin, Levo; levofloxacin, Novo; novobiocin.

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published 10 Feb, 2020

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Editorial decision: Minor revision
21 Jan, 2020
Review #1 received at journal
03 Jan, 2020
Submission checks completed at journal
21 Oct, 2019
Editor invited by journal
21 Oct, 2019
Reviewers invited by journal
21 Oct, 2019
Reviewer #1 agreed at journal
21 Oct, 2019
Editor assigned by journal
19 Oct, 2019
First submitted to journal
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You are reading this latest preprint version

Identification of Hemolysin encoding Genes and their Association with Antimicrobial Resistance Pattern among Clinical Isolates of Coagulase-Negative Staphylococci

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Journal Publication

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Abstract

Introduction

1-Methods

1–1-Identification of CoNS isolates

1–2-Antibiotic susceptibility testing

1–3-DNA extraction from isolates

1–4-Detection of hemolysin genes

1–5-Detection of mecA gene

1–6-Statistical analysis

2-Results

2–1-Isolation and prevalence of CoNS isolates

2–2-Antimicrobial susceptibility testing

2–3-Prevalence of hemolysin genes among CoNS isolates

2–4-Prevalence ofmecAgene among CoNS isolates

2–5-Statistical analysis

3-Discussion

Conclusion

Limitations

List of Abbreviations

Declarations

Ethical Approval and Consent to participate

Consent for publication

Availability of supporting data

Competing interests

Funding

Authors’ contributions

Acknowledgments

Authors’ Information:

References

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Supplementary Files

Status:

Journal Publication

Version 1