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Journal of Infection and Molecular Biology
Research Article
Inducible Clindamycin Resistance in Staphylococcus aureus Isolated from Palms of Poultry Workers in Jos, Plateau State, Nigeria
Ocheme Julius Okojokwu*1, Edoama Edet Akpakpan2, Hezekiah Yusuf Azi1, Bashiru Shafa Abubakar3, Joseph Aje Anejo-Okopi1
1Department of Microbiology, Faculty of Natural Sciences, University of Jos, Nigeria; 2Department of Biological Science, Federal University, Otuoke, Bayelsa State, Nigeria; 3Department of Zoology, Faculty of Natural Sciences, University of Jos, Nigeria.
Abstract | Background: Clindamycin is used for the treatment of infections attributed to macrolide (erythromycin) resistant Staphylococcus aureus; particularly infections of skin and soft tissues. Therapy for staphylococcal infections may be complicated by the possibility of inducible macrolide-lincosamide-streptogramin B resistance (iMLSB). Objective: This study was carried out to assess the prevalence of phenotypic expression of inducible clindamycin resistance of Staphylococcus aureus isolated from palms of poultry workers in Jos, Plateau State, Nigeria. Methods: A total of 186 Staphylococcus aureus were isolated and identified by conventional methods and subjected to antibiotic susceptibility testing by Kirby-Bauer disk diffusion method. Double disc approximation test (D-test) was used to investigate inducible and constitutive MLSB resistant phenotype. Results: From 186 S. aureus isolates, 113 (60.8%) were erythromycin resistant and 20 (10.8%) were clindamycin resistant. Most of the isolates 155 (83.3%) were methicillin-sensitive S. aureus (MSSA) while 31 (16.7%) were resistant to methicillin (MRSA). Out of the 186 isolates, 33 (17.7%) were iMLSB phenotype (D-test positive), 20 (10.8%) were constitutively resistant (cMLSB phenotype) and 60 (32.3%) were methicillin-sensitive (MS) phenotype (D-test negative). The incidence of constitutive and inducible clindamycin resistant phenotypes were higher in MRSA than MSSA. On the other hand, the incidence of MS phenotype was higher in MSSA than in MRSA. Conclusion: The study revealed that 17.7% of S. aureus were inducible clindamycin resistant which could have been misidentified as clindamycin susceptible by Kirby-Bauer disk diffusion method. The study also showed the importance of D-test in detecting inducible clindamycin resistance in S. aureus.
Keywords | Methicillin resistant, Staphylococci, Clindamycin, Inducible resistance, Constitutive resistance, D-test
Editor | Tahir Yaqub, University of Veterinary and Animal Sciences, Lahore, Pakistan.
Received | January 18, 2018; Accepted | February 11, 2018; Published | May 11, 2018
*Correspondence | Ocheme Julius Okojokwu, Department of Microbiology, Faculty of Natural Sciences, University of Jos, Nigeria; Email: okojokwuoj@gmail.com
Citation | Okojokwu OJ, Akpakpan EE, Azi HY, Abubakar BS, Anejo-Okopi JA (2018). Inducible clindamycin resistance in staphylococcus aureus isolated from palms of poultry workers in jos, plateau state, nigeria. J. Inf. Mol. Biol. 6(1): 11-15.
DOI | http://dx.doi.org/10.17582/journal.jimb/2018/6.1.11.15
ISSN (Online) | 2307-5465; ISSN (Print) | 2307-5716
Copyright © 2018 Okojokwu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread nosocomial pathogens (Harbath et al., 2005). The bacteria can be categorized as hospital-associated MRSA (HA-MRSA), community-associated MRSA (HA-MRSA) and livestock-associated MRSA (LA-MRSA) (Dahms et al., 2014). Close contacts to farm workers, especially family members, are at higher risk of acquiring MRSA (Dahms et al., 2014). Erythromycin (a macrolide) and clindamycin (a lincosamide) represent two distinct classes of antimicrobial agents of the MLSB family. Both of them bind to the50S ribosomal subunit thereby inhibiting protein synthesis (Yoon et al., 2008). The resistance to these two drugs can be mediated by msrAgene (MS phenotype) conferring the efflux mechanism (Ciraj et al., 2009; Deotale et al., 2010) or via the ermgene which encodes for the enzyme producing inducible or constitutive resistance to MLSB (Laclercq, 2002; Yoon et al., 2008). The resistance is constitutive (cMLSB) when R-methylase is produced and inducible (iMLSB) when methylase is produced only in the presence of an inducing agent.
To the best of our knowledge, no previous data on prevalence of drug resistance to erythromycin-clindamycin are available in Plateau State, Nigeria. This work therefore was done to close or address this knowledge gap. The aim of this work is to screen for LA-MRSA in poultry workers and to detect the prevalence of inducible clindamycin resistance among the S. aureus isolated from poultry workers.
MATERIALS AND METHODS
Study Area
This study was carried out in Jos (Jos South, North and East Local Government Areas) capital of Plateau State, North Central region of Nigeria. With about, nine hundred thousand (900,000) residents (NPC, 2006). It has a latitude of 9°56ʹN and longitude 8°53ʹE with monthly mean temperature of 21° – 25°C and 179km (111miles) from Abuja, the nation’s Federal Capital Territory (AGIS. 2010).
The study was a prospective study conducted during a period of 8 months (August 2015 to March 2016). A total of 186Staphylococcusaureusisolated from palms of poultry workers were used in the study. Staphylococcus aureus isolates were identified by standard biochemical techniques (Colle et al., 2006). Antimicrobial Susceptibility Testing (AST) was carried out for the coagulase positive S. aureus isolates using Kirby-Bauer disc diffusion method and interpreted as recommended by Clinical and Laboratory Standards Institute guidelines (CLSI, 2013). Antibiotic discs used were penicillin discs (10 units), cotrimoxazole discs (23.75/1.25 µg), cefoxitin discs (30 µg), cefuroxime discs (30 µg), gentamycin (10 µg), erythromycin discs (15 µg) and clindamycin (2 µg).
Isolates that were resistant to cefoxitin (30 µg) disc with zone of inhibition of inhibition ≤ 22 mm were taken to be methicillin resistant S. aureus (MRSA) while those with zones of inhibition were considered methicillin sensitive S. aureus (MSSA). Methicillin sensitive coagulase negative S. aureus (MRCoNS) were identified. All isolates were subjected to inducible clindamycin resistance testing by CLSI recommended D-test on Mueller Hinton agar by keeping erythromycin (15 µg) disc and clindamycin disc (2 µg) disc at 15 mm apart (edge to edge) (CLSI, 2013). Blunting of the circular zone of inhibition around clindamycin disc towards erythromycin disc indicated the presence of iMLSB resistance and was reported as resistance to clindamycin.
Following overnight incubation at 37°C, three different phenotypes were appreciated and interpreted as follows:
1. Constitutive MLSB phenotype: cMLSBS. aureus isolates which showed resistance to both erythromycin (zone size ≤13 mm) and clindamycin (zone size≤14 mm) with circular shape zone of inhibition around clindamycin.
2. Inducible MLSB phenotype: iMLSBS. aureus isolates which showed resistance to erythromycin (zone size≤13 mm) while being sensitive to clindamycin (zone size ≥21 mm) and giving D shaped zone of inhibition around clindamycin with flattening towards erythromycin disc (D test positive).
3. Methicillin-sensitive (MS) phenotype: S. aureus isolates exhibiting resistance to erythromycin (zone size ≤13 mm), while sensitive to clindamycin (zone size ≥21 mm) and giving circular zone of inhibition around clindamycin (no D zone i.e. D test negative).
Statistical Analysis
Statistical analysis was performed using Stata version 13, and P-values of ≤ 0.05 were considered statistically significant.
RESULTS
One hundred and eighty-six (186) staphylococcal isolates were obtained from palms of poultry workers, out of which 133 were resistant to erythromycin (87 methicillin susceptible S. aureus and 26 methicillin resistant S. aureus) while 20 S. aureua (15 methicillin susceptible S. aureus and 5 methicillin resistant S. aureus) were resistant to clindamycin (Table 1). One hundred and fifty-five [155(83.3%)] were susceptible to methicillin (MSSA) while 31(16.7) were methicillin resistant (MRSA). One hundred and thirteen [133(60.8%)] were found to be resistant to erythromycin; 20(10.8%) isolates were resistant to both erythromycin and clindamycin. Out of the 20 isolates, 15 isolates were methicillin sensitive S. aureus (MSSA) representing 9.7% of the MSSA while 5(16.1%) were methicillin resistant S. aureus (MRSA). This suggests constitutive macrolide-lincosamide-streptogramin B (cMLSB) phenotype. Thirty-three [MSSA: 20(12.9%); MRSA: 13(41.9)] isolates were positive to D-test which indicated inducible macrolide-lincosamide-streptogramin B (iMLSB) phenotype. Sixty-three [MSSA: 55(33.5%); MRSA: 8(25.8%)] isolates were negative to D-test indicating methicillin-sensitive (MS) phenotype. The isolates that were truly susceptible to clindamycin were 60. Relatively higher inducible and constitutive clindamycin resistance were observed in MRSA compared to MSSA (Table 2).
Table 3 depicts the relationship of D-test in relation to sex.
Table 1: Resistance profile of S. aureus to erythromycin and clindamycin
| Antimicrobial agent | MSSA | MRSA | Total |
| (n = 155) | (n = 31) | (n = 186) | |
| Erythromycin (15 µg) | 87 (56.1%) | 26 (83.9%) | 113 (60.8%) |
| Clindamycin (2 µg) | 15 (9.7%) | 5 (16.1%) | 20 (10.8%) |
| Total | 102 (65.8%) | 31 (100.0) | 133 (71.5%) |
MSSA = Methicillin sensitive Staphylococcus aureus, MRSA = Methicillin resistant Staphylococcus aureus
Table 2: Resistance phenotype of Staphylococcus aureus
| Total isolates |
Constitutive MLSB resistance (ERY-R, CLI-R) |
Inducible MLSB resistance (ERY-R, CLI-S, D+) |
MS phenotype (ERY-R, CLI-S, D-) |
|
| MSSA | 155(83.3%) | 15(9.7%) | 20 (12.9%) | 52 (33.5%) |
| MRSA | 31 (16.7%) | 5 (16.1%) | 13 (41.9%) | 8 (25.8%) |
| Total | 186 (100.0%) | 20 (10.8) | 33 (17.7) | 60 (32.3%) |
ERY-R: Erythromycin resistant (diameter of zone of inhibition ≤13mm); CLI-R: Clindamycin resistant (diameter of zone of inhibition ≤14mm); CLI-S: Clindamycin susceptible (diameter of zone of inhibition ≥21mm); MS: Methicillin sensitive; D+: D-shaped clear zone around CLI disc proximal to ERY disc; D-: Circular clear zone around CLI only; MLSB: Macrolide-lincosamide-streptogramin B.
Table 3: Chi square comparison of D-test positive and D-test negative S. aureus
| D-test | Isolates, n = 186 | Sex, n = 186 | MRSA, n = 31 | |||
| MSSA (%) | MRSA (%) | Male (%) | Female (%) | Male (%) | Female (%) | |
| D+ | 20 (60.6) | 13 (39.4) | 13 (39.4) | 20 (60.6) | 4 (30.8) | 9 (69.2) |
| D- | 135 (88.2) | 18 (11.8) | 59 (38.6) | 94 (61.4) | 7 (38.9) | 11 (61.1) |
| Total | 155 (83.3) | 31 (16.7) | 72 (38.7) | 114 (61.3) | 11 (35.5) |
20 (64.5) |
|
χ2 |
14.920 | 0.008 | 0.217 | |||
| p-value | < 0.001** | 0.929 | 0.641 | |||
| Association | Highly significant | Not significant | Not significant | |||
There was no statistically significant association (χ2 = 0.008; p = 0.929) between the response of the isolates to D-test and sex. Similarly, there was not association (χ2 = 0.217; p = 0.641) between the response of MRSA to D-test and sex. On the other hand, there was statistically significant association (χ2 = 14.920; p < 0.001) between D-test and type of S. aureus. Methicillin-sensitive S. aureus had higher D-test negative [135(88.2%)] isolates than MRSA [18(11.8%)]. In the same vain, more MSSA [20(60.6)] were positive than MRSA [13(39.4%).
DISCUSSION
This study established the existence of methicillin resistant Staphylococcus aureus on the hands of poultry farmers in Jos metropolis. Methicillin-resistant Staphylococcus aureus contamination of poultry products increases the emergence of antimicrobial resistance to humans. Poultry products are considered one of the main sources of spread of MRSA in humans. Commercial flocks, live bird markets, poultry litter, poultry slaughter houses and manure also play vital roles in propagation of resistant bacteria strain(s). This emphasizes the possibility of transmission between poultry birds (animals) and human and possibly from contact with pets at home. Staphylococcus aureus is one of the major pathogens that cause bacteremia and/or nosocomial infections. Antimicrobial resistance is an increasing problem in S. aureus infections all over the world (Tekin et al., 2013). The emergence of MRSA isolates led to difficulties for the treatment of infections caused by this microorganism. At the moment, up to 95% of clinical staphylococcal isolates are resistant to penicillin (Campanile et al., 2001; Sakoulas and Moellering, 2008). In the present study, methicillin resistance of S. aureus was found to be 16.7%. The difference in the prevalence of MRSA in different locations indicated that local antimicrobial testing has a significant role in empirical therapeutic decision making (Tekin et al., 2013). It is our opinion that 16.7% prevalence of MRSA in the poultry workers’ palm is high considering that these people were apparently healthy. It also suggests that beta-lactam antibiotics are inefficient agents for S. aureus in our communities hence it will be necessary to employ the use of different antibiotics for the treatment of infections caused by S. aureus.
Clindamycin is an alternative antibiotic for patients who are allergic to beta-lactam or who suffer from infections caused by MRSA. Clindamycin is indicated in the treatment of skin and soft-tissue infections caused by Staphylococcus species (Drinkovic et al., 2001; Nwokah and Abbey, 2016). Treatment failure can result when clindamycin or any non-inducer macrolide is used to treat infection caused by staphylococcal strain carrying inducible erm gene (Drinkovic et al., 2001). Therefore, in vitro test for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes which eventually results in treatment failure, thus the need to routinely screen for or detect such resistance by the double disk approximation test (D-test).
In this study, 60.8% of staphylococcal isolates were erythromycin resistant. Thirty-three (33 i.e. 17.7%) of the erythromycin resistant S. aureus isolates showed inducible clindamycin resistance. Some investigators have reported a higher incidence of iMLSB resistance while others indicated a lower incidence (Kumurya, 2015; Nwokah and Abbey, 2016). We observed, in our study, a similar incidence of iMLSB (17.7%) resistance among S. aureus and cMLSB (10.8%), even though a few others reported variable results. This difference or vairablity could be attributed to difference in geographical location, methicillin susceptibility of the S. aureus isolates and age group of the study subjects (Mohanansoundaram, 2011). The higher percentage of inducible clindamycin resistance in MRSA isolates (41.9%) as compared to MSSA (12.9%) is in concordance with various studies that reported the prevalence of erythromycin induced clindamycin resistance (Mohanansoundaram, 2011). We also observed that the MS phenotype was higher in the MSSA (33.5%) than MRSA (25.8%) which was in agreement with the findings of previously reported findings (Fiebelkorn et al., 2003; Fokas et al., 2005).
CONCLUSION
On account of emergence of resistance to antimicrobial agents among Staphylococcus aureus, the accurate antibiotic susceptibility data of the infecting bacteria is pertinent for making informed therapeutic decisions. On the whole, the inducible clindamycin resisant isolates obtained in our study was 17.7%. If D-test was not performed, those isolates would have been misidentified as being clindamycin susceptible thereby leading to therapeutic failure. Hand hygiene by poultry workers is therefore advocated to help reduce transfer of iMLSB Staphylococcus aureus from one person to the other.
ACKNOWLEDGEMENTS
The authors acknowledge the Department of Microbiology, Faculty of Natural Sciences, University of Jos, Nigeria for providing bench space in their facilities where the work was carried out.
CONFLICT OF INTEREST
There are no conflicts of interest.
AUTHORS CONTRIBUTION
Ocheme Julius Okojokwu conceived and designed the study, Ocheme Julius Okojokwu, Joseph AjeAnejo-Okopi, Hezekiah Yusuf Aziand Bashiru Shafa Abubakar performed the experiments; Ocheme Julius Okojokwu, Joseph AjeAnejo-Okopiand Bashiru Shafa Abubakar analyzed the data. Manuscript draft, reading, corrections and editing were done by Ocheme Julius Okojokwu, EdoamaEdetAkpakpan, Bashiru Shafa Abubakar and Hezekiah Yusuf Azi. All the authors consensually agreed to the final manuscript.
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