Antimicrobial susceptibility profile and research of mec A and erm genes in coagulase-negative staphylococci isolated from platelet concentrates bags

*Correspondence: Rosmari Horner. Laboratório de Bacteriologia. Departamento de Análises Clínicas e Toxicológicas. Prédio 26, Sala 1201 Campus da UFSM. Universidade Federal de Santa Maria 97015-900 Santa Maria RS, Brasil. E-mail: rosmari.ufsm@gmail.com Antimicrobial susceptibility profile and research of mecA and erm genes in coagulase-negative staphylococci isolated from platelet concentrates bags


INTRODUCTION
Bacterial contamination of blood components has probably been the first disease transmitted by transfusion (Blajchman, 1995).Despite bacterial sepsis associated to transfusion being known for a long time, it remains a serious health problem.Several measures have been deployed to prevent and detect bacterial contamination of platelet concentrates (PCs).These actions have reduced but not eliminated the risk of septic transfusion reactions (Eder, Goldman, 2011).Besides the risk of bacterial contamination of blood components, current studies seek to detect and reduce the risk of transfusion-related acute lung injury (TRALI), a syndrome characterized by acute respiratory distress following transfusion (Eder, Dy, O'Neill, 2016).
PCs are blood components with the highest bacterial contamination rate and usually responsible for most of septic transfusion reactions (Chang et al., 2004).This type of bacterial contamination due to PCs infusion is much higher than other types of transfusion-transmitted infections (Jacobs, Palavecino, Yomtovian, 2001;Goodnough, Sander, Brecher, 2003;Vasconcelos et al., 2004).
Gram-positive cocci are the most common contaminants in PCs and coagulase-negative staphylococci (CoNS) are the main responsible for septic transfusion reactions (Greco et al., 2008).They are frequently associated with hospital infections, particularly in immunocompromised patients (Stoll et al., 2002).
Although CoNS form part of the normal flora of humans, they are acknowledged as important pathogens, causing a wide variety of infections.They are isolated from skin and soft tissue infections, respiratory and lower urinary tract, and specifically bloodstream infections (Landrum et al., 2012).
In recent years, due to the high rate of hospital infections associated with antimicrobial resistance, resistance patterns of CoNS have become the study source of many researchers worldwide (De Giusti et al., 1999).
The classic mechanism of resistance to methicillin present in CoNS (coagulase-negative staphylococci resistant to methicillin -MRCoNS), encoded by the mecA gene is well documented, conferring resistance to all beta-lactam agents.Studies show that there has been a substantial increase in MRCoNS isolation rates in hospital infections, including Brazil, where resistance rates are among 50-80%, varying according to bacterial species and patient's demographic data (Marshall et al., 1998;Hussain et al., 2000;Ferreira et al., 2003;Palazzo, Darini, 2006).
CoNS may also be resistant to macrolides, lincosamides and streptogramins of group B agents (MLS B ), which may be constitutive (cMLS B ) or inducible (iMLS B ).In the constitutive expression, resistance to erythromycin and clindamycin is detected in a normal antibiogram.However, in the inducible form, resistance to erythromycin and false susceptibility to clindamycin are detected.It is recommended that the conventional phenotypic detection of iMLS B resistance is performed by D-test (Clinical and Laboratory Standards Institute, 2014).There are three mechanisms of Staphylococcus resistance linked to the MLS B group: target modification of ribosomes by methylase, mediated by erm genes (Nakajima, 1999); efflux mechanisms mediated by msrA gene (Matsuoka et al., 1999) and characterized as MS B phenotype, conferring resistance to macrolides only (Matsuoka et al., 2002;David, Pimentel, Freire, 2005); and antibiotics enzymatic inactivation (Roberts et al., 1999).Nevertheless, the predominant form is target modification (Roberts et al., 1999;Leclercq, 2002).
A study published by our research group emphasizes the importance of recognizing sepsis caused by contaminated PCs transfusion, specifically in immunocompromised patients, since bacterial sepsis associated to platelet transfusion is nowadays the most common infectious complication of transfusion medicine (Martini et al., 2012).
The aim of this study was to assess the susceptibility profile against antimicrobials as well as to research the typical resistance to methicillin and to MLS B group by phenotypic, conventional and automation methods, including mecA and erm genes.

Inclusion criteria for isolated bacterial and reference standard strains
A total of 16 CoNS were isolated through a study on bacterial investigation carried out in 691 PCs bags, collected during 2009/2010, from the Blood Bank of the city of Santa Maria, Rio Grande do Sul State (HEMORGS/ SM, RS), The following strains of international reference standard (American Type Culture Collection -ATCC) were used as controls in genotypic and phenotypic tests: Staphylococcus aureus ATCC 43300, ATCC 25923, BAA977, BAA976 and ATCC 29213.

Antimicrobial Susceptibility Testing by disk diffusion test (DD) and VITEK® 2; and determination of minimum inhibitory concentration (MIC) of vancomycin
For the performance of phenotypic tests, strains previously stored in Trypticase Soy Broth 15% glycerol were activated in Mueller Hinton agar and incubated at 35±2 °C for 24 hours.
We performed antimicrobial susceptibility testing using two methods, the conventional method of disk diffusion (DD) and the automated method VITEK® 2. Antimicrobial susceptibility testing was performed using disk diffusion test (DD) following the cutoff points recommended by the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2014), and susceptibility testing was performed by VITEK® 2 automated method, according to the manufacturer's standards (bioMérieux).Vancomycin minimum inhibitory concentration (MIC) was made by the broth micro-dilution method, performed in duplicate (Clinical and Laboratory Standards Institute, 2014).

Analysis of resistance genes
For the genotypic testing, deoxyribonucleic acid extraction (DNA) was carried out by heating shock.Bacterial isolates were grown on Trypticase Soy Agar and then incubated at 35 ± 2°C for 18 to 24 hours.Subsequently, a dense bacterial suspension with 4-5 isolated colonies was prepared in an Eppendorff type microtube containing 500 uL autoclaved Milli Q water.This suspension was homogenized by vortex and placed in water bath at 100 °C for 15 minutes.The microtube was subjected to heating shock (on ice) for 5 minutes and centrifuged at 11,000 rotations per minute (rpm) for 20 minutes.The supernatant (containing the DNA) was transferred to another microtube which was frozen at -20 °C (Nogueira et al., 2004;Freschi, Carvalho, Oliveira, 2005;Scheffer, 2008;Aguilar, 2009).DNA extraction by heating shock was confirmed by the 16S rRNA gene amplification, obtaining a high quality DNA.

Amplification of 16S ribosomal RNA (rRNA)
The extracted DNA was subjected to electrophoresis in 1.0% agarose gel to verify DNA presence in the extracted material.The 16S rRNA was amplified by Polymerase Chain Reaction (PCR) and the following primers were used: 5'-GTAGTCCACGCCGTAAAC-3' and 5'-TAAACCACATGCTCCACC-3 (Li et al., 2009).

Identification of MRCoNS
Identification of bacterial species isolated from infected PCs bags was performed by VITEK® 2 automated system.To study phenotypic resistance to methicillin were used discs of cefoxitin (30 µg) and MIC cefoxitin (VITEK® 2).Results were interpreted according to the criteria established by the CLSI (Clinical and Laboratory Standards Institute, 2014).
To identify the presence of mecA gene, the following sequences were used: mecA_F AGT TAG ATT GGG ATC ATA GCG and mecA_R CGA TGC CTA TCT CAT ATG CTG (Ludwig Biotec®).The primers used for mecA gene testing were designed using the PBP2a protein structure made in the Laboratory of Molecular Biology and Sequencing -LabDros (Lim, Strynadka, 2002).The best mold region for primer design is the non-variable portion of PBP2a -between 27 and 326 amino acids -being in base pairs, corresponding to the region between 81 and 978 nucleotides.The GU301106 reference sequence from GenBank was used as the primer's choice.The size of amplicons generated by the amplification using these primers is 145 bp, and they were checked in 0.8% agarose gel.MRCoNS identification was followed as suggested by Kearns et al. (1999).

Statistical analysis
We proceeded to sensitivity assessment and methods specificity tested in the detection of each resistance mechanism (Bonita, Beaglehole, Kjellström, 2010).

Ethical considerations
This study was approved by the Research Ethics Committee (CEP) of UFSM under the number 0285.0.243.000-09.

Automated bacterial identification -VITEK® 2
From the total of 16 isolates of CoNS obtained through a research on bacterial contamination carried out in 691 PCs from HEMORGS/SM, five were identified as Staphylococcus haemolyticus (31.25%), four as Staphylococcus epidermidis (25%), four as Staphylococcus warneri (25%), and three as Staphylococcus saprophyticus (18.75%).The bacterial species isolated from contaminated PCs bags were identified by the automated system Vitek® 2. All organisms isolated in contaminated PCs were identified as CoNS, similar to the results found by several authors (Hsueh et al., 2008;Martínez, Tarrand, Lichtiger, 2010;Walther-Wenke et al., 2010).Currently, CoNS represent one of the major nosocomial pathogens and S. epidermidis and S. haemolyticus are the most significant species.Moreover, the increasing rates of antibiotic resistance are an even greater problem for CoNS than for Staphylococcus aureus, limiting our therapeutic options (Becker, Heilmann, Peters, 2014).

Comparison of susceptibility profile between disk diffusion test (DD) and VITEK® 2
In this study, there was discrepancy in the susceptibility assessment between DD and automation methods with clindamycin, sulfamethoxazole/ trimethoprim, erythromycin, gentamicin and norfloxacin antibiotics (in descending order), and agreement was observed only in three (No. 11,12 and 14) of the 16 analyzed samples as they showed the same profile of antimicrobial susceptibility (Table I).
Studies by other authors confirm our findings when these two methods are compared (VITEK® 2 and DD test -considered reference methods), reporting the occurrence of much higher error (VME), higher error (ME), and lowest error (MiE) with the VME prevalence profile (Sapino et al., 2012;Paim, Cantarelli, D'Azevedo, 2014), in susceptibility assessed by the automated method.
All CoNS were resistant to penicillin G and penicillin (automated and DD test, respectively), and by DD test 62.5% were resistant to clindamycin and erythromycin against 31.25% and 43.75%, respectively, by automation.Studies have reported CoNS high resistance levels to penicillin, clindamycin and erythromycin (Bernardi, Pizzolitto, Pizzolitto, 2007;Melo et al., 2007;Rigatti et al., 2010).All samples were susceptible to vancomycin, a similar result to that found in the studies mentioned above.In our study vancomycin MIC was between 0.5 µg/mL a 2 µg/mL.

Assessment of Methicillin-Resistant Coagulase-Negative Staphylococci (MRCoNS)
In relation to methicillin resistance in CoNS, three samples (18.75%) were resistant to cefoxitin in DD, and by VITEK® 2 one sample was resistant (Table II).Regarding CoNS, the standardized tests reporting MRCoNS strains are: agar dilution, DD technique and broth micro-dilution (Clinical and Laboratory Standards Institute, 2014).In the study developed by John et al. (2009), authors showed that, VITEK® 2 did not detect methicillin resistance against S. aureus and most of the CoNS.By the PCR method, considered by many authors the gold standard one for the phenotypic testing comparison (Cafiso et al., 2004;Ruzicka et al., 2004;Gad et al., 2009), the mecA gene presence was detected in three samples (18.75 %), which were susceptible to cefoxitin in DD test and VITEK ® 2 (Table II).
Studies show that methicillin resistance in Staphylococcus occurs mainly due to the production of modified penicillin binding protein (PBP2a) encoded by the mecA gene.This less frequent resistance, however, may also be due to the beta-lactamase overproduction by oxacillin inactivation, resulting in partial hydrolysis of the beta-lactamase ring (borderline resistance to methicillin -BORSA) or the presence of a PBP with low affinity to oxacillin (MODSA) (Tomasz et al., 1989;Ito et al., 2003;Rossi, Andreazzi, 2005).In these last two resistance mechanisms to methicillin, bacteria do not carry the mecA gene (Mohanasoundaram, Lalitha, 2008).
We suggest the identification of three MRCoNS strains with overproduction of beta-lactamase enzyme, which rarely occurs when Staphylococcus strains show resistance in DD test and do not have the mecA gene.The treatment for infections caused by MRCoNS may be performed with linezolid, teicoplanin, and vancomycin, as well as alternative drugs such as macrolides, clindamycin, and trimethoprim/sulfamethoxazole (Rossi, Andreazzi, 2005).
Resistance to methicillin and beta-lactamase overproduction are the main causes of discordant results among phenotypic and genotypic tests to detect MRCoNS (Ghoshal et al., 2004).Research of this kind of resistance by phenotypic methods can be complex due to the heterogeneous expression of many strains, which may also be influenced by growth conditions (Chambers, 1997).
Recent surveys have detected a rare homologous gene to mecA in S. aureus strains isolated from humans and animals with phenotypic resistance to methicillin, found through DD test with cefoxitin, which did not show the mecA gene when specific primers for this gene were used (Cuny et al., 2011;Shore et al., 2011;Cartwright et al., 2013).This gene was initially called mecA LGA251 and later designated as mecC (Ito et al., 2012).This mecC gene has also been reported in CoNS as Staphylococcus stepanovicii, Staphylococcus xylosus and Staphylcoccus scirui (Harrison et al., 2013a(Harrison et al., , 2013b;;Loncaric et al., 2013).As already suggested for the mecA gene, we can probably infer that the origin of the mecC gene is also among the CoNS (Tsubakishita et al., 2010;Couto et al., 2013).Although mecC gene detection is rare and reported only in Europe so far, this gene is a potential diagnosis problem and triggers several issues for future studies (Paterson, Harrison, Holmes, 2014).

Analysis of macrolide-lincosamide-streptogramin B (MLS B ) resistance
The conventional phenotypic analysis (DD) of resistance to MLS B antimicrobials, carried out with the disk approximation test -D-test, characterized six samples (37.5%) with inducible (iMLS B ) and two samples (12.5%) with constitutive phenotype (cMLS B ).On the other hand, the MS B phenotype was not detected by DD test (Table III).Coutinho et al. (2010) analysis of CoNS through the DD method found three different phenotypes, distributed as follows: 46.7% cMLS B and 3.3% of the other two phenotypes (iMLS B and MS B ). Through the automated method, our study detected 31.25% (5 of 16) iMLS B and 6.25% (1 of 16) MS B (Table III).As mentioned in the paragraph above, no CoNS of our research showed an efflux mechanism (MS B phenotype) by the D-test result.However, when automation was used (VITEK® 2) one sample showed the MS B phenotype: this one showed the iMLS B phenotype by DD test.
The erm gene was detected in five PCR samples (31.25%).One (6.25%) containing ermA gene and four (25%) ermC gene; the ermB gene was not detected in our study.Coutinho et al. (2010) identified the presence of ermA gene in 29.6% of samples.Regarding the genotypic test, our index showed 67% of sensitivity and 86% of specificity, when compared to D-test, which was considered a reference in this study.Lavallée et al. (2010) compared detection methodologies for iMLS B resistance only, in a collection of 163 Staphylococcus with resistant or intermediate profile to erythromycin and susceptible profile to clindamycin.In our study, through the performance of the same phenotypic tests and erm genes detection by PCR, 93% and 100% specificity and sensitivity were found, respectively, for VITEK® 2. Therefore, they concluded that automation has failed to detect iMLS B resistance in ten strains (10/163), resulting in a negative predictive value (NPV) of 74% (Lavallée et al., 2010).When comparing these same methodologies, in our study we found 73% of NPV.
The authors cited above concluded that D-test is the most simple and reliable technique to detect this resistance in CoNS strains.They also concluded that agar dilution and VITEK® 2 are not as sensitive as D-test within 24 hours for CoNS strains for the detection of iMLS B (Lavallée et al., 2010).
The discrepancy between results given by the different methods, i.e., inhibition in DD test and gene absence in PCR may be justified by the presence of another gene, ermY, which was not the subject of this research.However, there are reports of its presence in Staphylococcus (Roberts et al., 1999).The other reason for strains showing the iMLS B phenotype that do not contain the erm gene is still not clear (Roberts et al., 1999), but it is attributed to possible changes in the local targets of primers for these genes (Lavallée et al., 2010).Our study confirmed the superiority of D-test when compared to VITEK®2 in iMLS B detection.

CONCLUSIONS
As for resistance genes showed by the different species studied, it was found that one S. epidermidis and one S. warneri showed the mecA gene and the erm gene.These two species also showed the highest resistance rate to methicillin.Through the antimicrobial susceptibility profile, obtained by the DD technique, approximately 50% of strains were characterized as multi-drug resistant (MDR), being resistant to more than three classes of antimicrobials.
The results of this study are a reminder to health professionals regarding the CoNS antimicrobial susceptibility profile, since most of them were resistant to antimicrobials (A and B-CLSI ratings) by the conventional assessment method.Also, half of the isolates were resistant to multiple classes of antibiotics, which directly influence on morbidity and mortality, specifically in immunocompromised patients, who often develop infections from resident microorganisms of the skin flora only with strains that showed phenotypic resistance to methicillin; CFO = Cefoxitin; DD = Qualitative methodology of disk diffusion; S = Susceptible; I = Intermediate; R = Resistant; "-"= absence; "+"= presence.such as CoNS.Restrictive policy with antimicrobials as well as immediate and correct prescription should be encouraged in order to control bacterial resistance.This is in fact the first study conducted in Brazil that investigates susceptibility profile and resistance mechanisms of microorganisms isolated from PCs bags.Bras. Anal. Clin., v.39, p.115-118, 2007.

TABLE I -
Comparison of susceptibility profile of the 16 isolates of coagulase-negative staphylococci from Platelet Concentrates, using the qualitative methodologies of Disk Diffusion Test (DD) and VITEK ® 2, according to the criteria of CLSI 14

TABLE II -
Analysis of MRCoNS of the 16 coagulase-negative staphylococci isolates from Platelet Concentrates through the phenotypic tests of Cefoxitin Disk Diffusion (DD), screening of cefoxitin by VITEK ® 2, research of the gene mecA by Polymerase Chain Reaction