Inducible clindamycin-resistant and biofilm formation in the Staphylococcus aureus isolated from healthcare worker’s anterior nasal carriage

Objective The purpose of this study is a new update on the resistance profile, Macrolide–Lincosamide–Streptogramin B resistance mechanisms and biofilm formation in the Staphylococcus aureus isolated from health care workers (HCWs) nasal carriage at a children’s teaching hospital in Babol (Northern Iran). Results A total of 143 non-repetitive nasal swab samples were collected from volunteers, where 53.8% (n; 77/143) were HCWs, 33.6% (n; 48/143) medical students, and 12.6% (n; 18/143) resident students. The prevalence of nasal carriers of S. aureus was 22.4% (n; 32/143), among them, 40.6% (n; 13/32) were identified as methicillin-resistant Staphylococcus aureus (MRSA( carriers. Antimicrobial susceptibility testing showed that erythromycin (68.8%, n; 22/32) and ciprofloxacin (15.6%, n; 5/32) had the highest and lowest resistance rate, respectively. The frequency of resistance genes in the strains was as follows; ermC (n; 17/32, 53.1%), ermA (n; 11/32, 34.4%), ermB (n; 6/32, 18.7%), ereA (n; 3/32, 9.4%). Moreover, 50.0% (n; 16/32), 28.1% (n; 9/32) and 21.8% (n; 7/32) of isolates were strongly, weakly and moderately biofilm producer, respectively. Macrolides-lincosamides-streptogramins B (MLSB) antibiotic resistance among S. aureus isolates from HCWs nasal carriage have found significant prevalence rates throughout the globe. It is crucial to remember that the development of biofilms and MLS B antibiotic resistance are both dynamic processes.


Introduction
Staphylococcus aureus, a catalase-and coagulase Grampositive cocci is a prominent pathogenic microorganism that can lead to a multiple infections from minor skin and soft tissue infections (SSTIs) to severe and potentially fatal diseases [1].The nostril is the most common carriage site for S. aureus and anterior nasal carriers are at high risk of developing S. aureus infections.Human colonization with S. aureus occurs in the first days of life [2].Nasal carriers are divided into transient and permanent.Antibiotic resistance and biofilm formation are important factors in maintaining the carrier state [3].
Macrolide, lincosamide and streptogramin B (MLS B ) are effective as a limited and alternative treatment regimen in Staphylococcal infections, especially in SSTIs.Multiple mechanisms have been identified that confer resistance to MLS B antibiotics.These mechanisms include the presence of an active efflux pump encoded by the msr gene, drug inactivation by the lun gene, and the presence of the erm cluster, which induces changes in the ribosomal binding site via methylation and/or point mutation [4][5][6][7].The msrA gene has been found to be present in S. aureus and is responsible for the ATPdependent transport of erythromycin and streptogramin B out of the cell [6].Also, msr plasmid genes encoding macrolide efflux pump have been described in these bacteria [6].
Biofilm, an extracellular polysaccharide matrix that surrounds bacteria, is one of the important survival and resistance factors in the carriage.The presence of polysaccharide intercellular adhesive (PIA) encoded and regulated by the intercellular adhesion operon (ica ADCB) is essential in biofilm formation [8].The operon consists of three components: a N-acetylglucosamine transferase (icaA and icaB), a predicted exporter (icaC), and a deacetylase (icaD).
Understanding the mechanisms of antimicrobial resistance and biofilm formation in S. aureus nasal carriers can offer valuable insights for enhancing infection control measures and improving clinical treatment strategies in the future [9].Therefore, the purpose of this study is to provide a new update on the resistance profile, MLSB resistance mechanisms, and biofilm formation in S. aureus isolated from health care workers (HCWs) nasal carriage at a children's teaching hospital in Babol, Northern Iran.

Study design, sampling and laboratory identification
The cross-sectional study was performed with the committee ethical number of IR.MUBABOL.HRI.REC.1400.159from the one-year period of time (2022) at the Amirkola children's teaching hospital (Babol, north of Iran).Exclusion criteria was HCWs who received antibiotics for the previous two weeks or those suffering signs and symptoms of upper respiratory tract infections.

Collection and processing of nasal swabs
A single nasal sample was obtained from each participant, by gently inserting a swab into their nostril and rotating it three times.The swabs were then transported to the laboratory under sterile conditions.Following this, the samples underwent culturing on Mannitol agar that had been supplemented with 7.5% sodium chloride (Merck Co., Germany), and were then incubated for a period of 24 h at a temperature of 37˚C.Standard microbiological and biochemical methods were employed to identify all resulting colonies.PCR of nuc gene (encoding thermonuclease) was used to confirm S. aureus strains [4,10].

Determination of inducible resistant phenotypes
To identify resistant phenotypes, a double disk test was conducted by placing ERY and CD disks 20 mm apart as previously described [11].

Crystal violet biofilm formation assay
Biofilm production ability was assessed using 96-well flat bottom microtiter plate procedure as previously described.

Molecular detection of resistance determinants
Bacterial cells were lysed as follows: five pure colonies liquefied in a 25 µl of 0.25% sodium dodecyl sulfate (SDS)-0.05N NaOH solutions and heated for 15 min.After adding 200 µL of ddH2O to the microtube, 5 µL of the diluted mixture was used in the PCR method.Successful DNA isolation was verified via agarose gel electrophoresis.Multiplex-PCR assay was performed by DNA amplification device (Eppendorf, Germany) to detect the icaA, icaB.icaD, ermA, ermC ,ereA, msrA, msrB using the specific primers (Table 1) [4,12].
PCRs were conducted in an Eppendorf Co. (Germany) master cycler gradient, with a final reaction volume of 25 µl composed of 2.5 µl of template DNA, 13.5 µl of Taq DNA Polymerase Master Mix RED (Ampliqon, Stenhuggervej, Odense M, Denmark), 1.0 µl of each primer, and 7.0 µl of ddH 2 O water.

Statistical analysis
Statistical analysis in this study was carried out with SPSS software version 22.0 (IBM, Armonk, NY, USA), and the chi-square test was utilized to compare the data related to biofilm formation and resistance genes.A significance level of less than 0.05 was considered statistically significant.
The data indicated that 34.4% (n = 11/32) of the isolates demonstrated resistance to both CD and ERY.

22 msrA F;5ʹ -T C C A A T C A T T G C A C A A A A T C-3ʹ
52.7 20 163

R;5ʹ -A A T T C C C T C T A T T T G G T G G T-3ʹ
53.8 20

msrB F;5ʹ -T A T G A T A T C C A T A A T A A T T A T C C A-3ʹ
48. 4

595 R;5ʹ -A A G T T A T A T C A T G A A T A G A T T G T C C-3ʹ
52.8 25

26 880 F;5ʹ -T C T A A T C T T T T T C A T G G A A T C C G T-3ʹ
56.4 24 60.6 24 1066

F;5ʹ -T A A T A A G C A T T A A T G T T C A A T T-3ʹ
47.8 22

20 198 R: 5'-A G T A T T T T C A A T G T T T A A A G C A A A T A C-3ʹ
54. 4 27 Specifically, 9.4% (n = 3/32) of the strains displayed a resistant phenotype to cMLSB (i.e., resistant to both ERY and CD), 18.8% (n = 6/32) showed inducible resistance iMLSB (i.e., resistant to ERY but susceptible to CD), and 6.3% (n = 2/32) of the isolates had the MS phenotype (i.e., susceptible to ERY and resistant to CD).

Discussion
In fact, about 20-30% of humans can carry this organism continuously and asymptomatically.Therefore, nasal carriers can increase the risk of infection transmission, which leads to the serious infections, especially in hospitalized patients and immunocompromised, which is linked to increased risk of death and prolonged hospital stays [13][14][15].In the present study, the prevalence of  [18].These differences can be the result of the study population, the place of sampling (specialized hospital compared to general hospitals) and people's awareness of personal medical-hygiene.The rate of colonization was significantly higher in people who did not use antibiotics at least in the last 3 months (0.03 and 0.05 for MSSA and MRSA, respectively).Significantly, colonization was more in people who were in contact with the patient, which suggests an increase in the incidence of iatrogenic disease.The highest colonization rates of MSSA and MRSA were respectively in nurses (42.1% and 38.5%) and then medical students (21.1% and 23.1%).In a contrast study at the Brazil, Danelli et al.. (2020) demonstrated that males and students had a significantly higher prevalence of S. aureus carriage (OR = 2.898);However, no factors were found to be correlated with the carriage of MRSA [17].
However, the observed differences in S. aureus and MRSA carriage rate in the country and other parts of the world can be attributed to variations in sample size, identification methods and local infection control polices [22,23].
On the other hand, several probable factors contribute to the high prevalence of MRSA among HCWs.These include inadequate cleaning and disinfection protocols, high patient-to-staff ratios that may result in lapses in hygiene practices, and the frequent interaction of HCWs with patients who are positive for MRSA as opposed to those who are negative for MRSA, particularly in intensive care units or during medical procedures [24,25].
Contrary to our study, Omidi et al., (2020) showed that 76.0% (n; 111/146) and 87.5% (n; 21/24) of S. aureus and MRSA strains were able to strong biofilm production, respectively.75% (n = 18/24) of MRSA isolates tested were found to possess the icaA gene, whereas no icaD gene was detected [34].This difference can be due to the presence of genes other than ica that play a role in biofilm formation.Biofilm formation by S. aureus has been suggested to be primarily driven by the PIA pathway, encoded by the ica operon.However, there is evidence of an ica-independent pathway, linked to the expression of Bap [35].In addition, it has been observed that methicillin resistance is linked to the inhibition of PIA and biofilm formation dependent on surface proteins.

Conclusion
Our findings indicate a considerable prevalence of MRSA colonization among HCWs, highlighting a persistent and significant healthcare challenge within our region.Conversely, the management of antibiotic prescriptions to mitigate selective pressures is essential for addressing the emergence of multidrug-resistant (MDR) isolates, including vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) strains.To enhance the management of S. aureus infections, techniques for avoiding biofilm development and dissolving existing biofilms should be investigated.

Limitations
This study is subject to certain limitations: the primary limitation pertains to the incomplete availability of comprehensive patient history background information.Furthermore, it is imperative to acknowledge that the isolation of MRSA strains was confined to different hospitals, necessitating a cautious approach to the interpretation of the results.

Table 1
The primer sequences used in this study

Table 2
The possible risk factors associated with the nasal carriage of MSSA and MRSA among the study participants