Bacteriologic and plasmid analysis of etiologic agents of conjunctivitis in Lagos, Nigeria

The infections of the eye including conjunctivitis are a major cause of hospital consultation in Nigeria and are associated with hospitalization and mortality rates of 0.3–3% [22–24]. The present study shows that conjunctivitis in Lagos is caused by multiple bacterial aetiologies involving both Gram-positive and Gram-negative pathogens with varying antibiotic susceptibility patterns and plasmid carriage ability. The transfer of antibiotic resistance and R plasmids by some of the recovered strains to E. coli was also revealed. The observed etiologic pattern of Gram-positive pathogens predominated by S. aureus (27.7%) and non-coagulase Staphylococci (22.6%) as causative agents of conjunctivitis in this study is similar to previous reports by Iroha et al. [6] who reported 37.4% and 12.3% as the isolation rates for S. aureus and coagulase-negative Staphylococci in the same environment. In southeast Nigeria, Ubani [25] also recovered S. aureus (23.7%) and Staphylococcus albus (19.3%) as the predominant Gram-positive pathogens of ocular infections in patients. In other countries of the world such as India, isolation rates of 25% and 18.3% have been reported in S. aureus and non-coagulase Staphylococci as ocular pathogens, respectively [8]. Generally, the predominance of Staphylococci, Bacillus sp., and Corynebacterium sp. as major ocular pathogens might be due to the fact that these organisms represent the major flora of the eye lid and the conjunctiva and under normal conditions, their clinical manifestations are averted by eye innate immune defense system constituted by tear flow, secretory immunoglobulin, and the presence of cidal agents such as lysozyme and lactoferrin [9, 26]. Other Gram-positive pathogens such as S. pneumoniae, S. pyogenes, S. viridans, and Haemophilus influenzae were not found in this study and were not reported by Iroha et al. [6]. This suggests consistency in the Gram-positive etiology of conjunctivitis in Lagos, even though non-coagulase Staphylococci are now trending toward equality with S. aureus in causing conjunctivitis in the study area. Contrastingly, these pathogens were implicated as ocular pathogens in southeast Nigeria [25], India [8], Hong Kong [27], USA [12], Singapore [9], and Ghana [10], reconfirming previous reports that ocular pathogens vary in etiology in different countries and different locations within a country [8, 25, 28]. Our non-recovery of other Gram-positive ocular pathogens may also be due to the focus of this study, which centered on conjunctivitis compared with other ocular infections included in the Indian and southeastern Nigerian studies [8, 25]. With regards to Gram-negative pathogens in ocular infections in Lagos, we found P. aeruginosa as the predominating strain with an isolation rate of 9.7%, whereas in a previous study in the same environment, Klebsiella pneumoniae was the most commonly isolated pathogen (12.9%) followed by P. aeruginosa (8.2%). Therefore, our finding indicates a changing trend in the Gram-negative etiology of conjunctivitis in Lagos. In other parts of the country, K. pneumoniae was also most commonly isolated, followed by E. coli coupled with the involvement of other Gram-negative pathogens such as N. gonorrhoeae and Neisseria meningitides. This again corroborates the influence of locations on the etiology of ocular infections. Unlike the present study, which is similar in context to the study in southeast Nigeria, the previous Lagos study focused on neonates who have predilections to infections due to E. coli and Klebsiella sp. in that age group. In the present study, the 83 conjunctival samples tested were obtained from patients of all age groups, with 10.8% being children aged 2–5 years, 27.7% were aged 6–13 years representing older children, 21.7% were adolescents aged 14–19 years, and 39.8% were adults.

Our results of pathogens’ antibiotic susceptibilities suggest that in vitro, chloramphenicol has deteriorated as an anti-conjunctival agent with an overall efficacy of 63.9% and being 55.8–90% and 20–71.4% effective against Gram-positive and Gram-negative bacteria isolated, respectively. In an increasing order anti-conjunctival activity, other tested antibiotics’ activities are streptomycin < ampicillin < gentamicin < amoxicillin < cefotaxime < amikacin < ceftazidime < ofloxacin, with efficacy ranging from 77.4% to 96.1%. In clinical context, our findings connote a possible reduction in the therapeutic relevance of chloramphenicol and gentamicin in the treatment of conjunctivitis in Lagos as these antibiotics in the form of eye drops are most commonly available and used in the study area. Given the higher anti-conjunctival efficacies of amikacin, cefotaxime, and ofloxacin, the availability as eye preparations and the empirical use of these antibiotics are strongly recommended in the management of conjunctivitis in Lagos. Elsewhere, fluoroquinolones such as ofloxacin and gatifloxacin are also the current recommended drugs of choice in the treatment of ocular infections [8–10, 29, 30]. However, to prevent and control the development of antibiotic resistance by ocular pathogens to these antibiotics and even optimize the therapeutic applications of chloramphenicol and gentamicin, institution of therapy based on the antibiogram profile of pathogens is highly essential. Furthermore, our observation that 69.9% of conjunctivitis cases screened are polymicrobial in etiology provides an indication for a complex epidemiological situation of conjunctivitis in this environment. Therefore, institution of appropriate anti-conjunctival therapy in afflicted patients is very important as this would avert further complications and improve the prognosis of infection. Based on our findings, such appropriateness in medication may warrant the use of two or more antibiotics especially for cases in which both Gram-positive and Gram-negative pathogens are involved.

The present study has also documented the involvement of plasmids as factors responsible for antibiotic resistance in some of the recovered pathogens since these resistances were partly transferred to E. coli DH5α by conjugation. Therefore, our findings suggest the emergence and active transfer of antibiotic resistance and R plasmids among the circulating strains causing conjunctivitis in Lagos. In this study, antibiotics such as chloramphenicol, streptomycin, and ampicillin were easily transferred from a multidrug-resistant ocular pathogen to E. coli. The co-transfer of plasmids of sizes 21.5, 15.2, and 5.0 kb suggests that these extrachromosomal DNAs are R plasmids, even though curing experiment was not done. The minimum size of a plasmid with an efficient conjugation system has been reported to be >15 kb [14]. Therefore, the presence of the 5.0-kb plasmids in the transconjugants implies that the larger molecular size plasmids (i.e., 21.3 and 15.2 kb) might serve as vehicles for the transfer of lower molecular weight plasmids such as 5.0 kb to E. coli DH5α. This also implies that the mechanism of plasmid mobilization among the ocular pathogens may entail the use of larger sized plasmids as vehicles in addition to the classical transfer systems such as conjugation that require considerable genetic information, as previously reported by Smith and Linggood [31], Achmith and Helmuth [32], Christiansen et al. [33], and Jamieson and Bremner [14]. The result from the conjugation experiment also revealed that the varied drug-resistant exconjugants arose at a frequency range of 10⁻⁵–10⁻⁷ per donor cell. These evolution rates are similar to those of previously reported epidemiological and clinically important pathogens in Nigeria, and some of these pathogens were also recovered in this study. They include P. aeruginosa [34], E. coli [35], and Staphylococci [36]. Of further clinical importance is the fact that the organisms recovered here have also been implicated in the pathogenesis of other non-ocular infections such as diarrhea illnesses, urinary tract infections, sepsis, bacteremia, pneumonia, and meningitis [37–39]. In these infections, resistance rates higher than what we found for the least and most active anti-conjunctival antibiotics in this study were reported [38, 39]. Therefore, our antibiogram results indicate that the conjunctivitis cases studied bacteriologically are probably exogenous in origin. This possibility is further supported by the fact that all the patients studied are outpatients ruling out nosocomial ocular infections. The prevalence rate of plasmids of 44.6% can be said to be high, and this is synonymous with pathogens that elicit resistance to two or more antibiotics. Lower plasmid prevalence rates have been reported for pathogens such as P. aeruginosa [40], while higher rates have been reported for multidrug-resistant strains of E. coli and K. pneumoniae in epidemiological studies [41]. In this study, plasmids having a size range of 5.0–12.5 kb were common among the Gram-negative pathogens and those of 2.7–4.1 kb were common among the Gram-positive pathogens irrespective of the species affiliations, suggesting genetic diversity disparity but yet active transfer of plasmids and associated antibiotic resistances among the ocular pathogens in causing polymicrobial infections in patients with conjunctivitis in Lagos.

The implications of the results obtained from this study are twofold. First, the epidemiological situation of conjunctivitis is more complex than previously thought in this environment. Secondly, the etiologic agents harboring plasmids have the potentials of disseminating antibiotic resistance and their associated plasmids to other causative agents without plasmids that were previously sensitive to the commonly used anti-conjunctival agents in Lagos. To avert these implications, there is a need to carry out regular surveillance of agents of conjunctivitis in Lagos for antibiotic susceptibility and plasmid carriage in order to develop an appropriate strategy to prevent the spread of antibiotic resistance among pathogens and control conjunctivitis. Further studies are required using, apart from plasmid curing, techniques such as polymerase chain reaction and drug resistance phenotyping methods for better understanding of mechanisms to antibiotic resistance by Gram-positive and Gram-negative pathogens responsible for conjunctivitis and other ocular infections in Nigeria. Studies are also needed to understand the pathogenic disposition of some of these isolates, vis-a-vis the virulent factors they express using both in vitro and in vivo model systems. This is because the conjunctiva and cornea sac for instance naturally accommodate avirulent microbes as flora, and these organisms can be co-recovered from clinical eye specimens in patients with eye problems [9, 26].

Nevertheless, based on the findings of this study, it can be concluded that conjunctivitis as an eye problem in Lagos is polymicrobial with infections eliciting low and high sensitivity to chloramphenicol and ofloxacin, respectively. Infections are also associated with transferable R plasmids for chloramphenicol, ampicillin, and streptomycin. Continuous surveillance of conjunctivitis in relation to etiology, drug susceptibility, plasmid transferability, and mechanisms of resistance for epidemiological control in the study area is therefore recommended.