Trends in aetiology and antibiotic resistance in bacterial keratitis isolates from South India between 2013-2024

Trends in aetiology and antibiotic resistance in bacterial keratitis isolates from South India between 2013-2024 | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Trends in aetiology and antibiotic resistance in bacterial keratitis isolates from South India between 2013-2024 View ORCID Profile Prajna Lalitha , View ORCID Profile Rameshkumar Gunasekaran , View ORCID Profile Leonie Fingerhut , View ORCID Profile Namperumalsamy Venkatesh Prajna , View ORCID Profile Bethany Mills doi: https://doi.org/10.1101/2025.10.01.25337045 Prajna Lalitha 1 Department of Ocular Microbiology, Aravind Eye Care System , Madurai, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Prajna Lalitha For correspondence: lalitha{at}aravind.org beth.mills{at}ed.ac.uk Rameshkumar Gunasekaran 1 Department of Ocular Microbiology, Aravind Eye Care System , Madurai, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rameshkumar Gunasekaran Leonie Fingerhut 2 Institute for Regeneration and Repair, The University of Edinburgh , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Leonie Fingerhut Namperumalsamy Venkatesh Prajna 3 Department of Cornea, Aravind Eye Care System , Madurai, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Namperumalsamy Venkatesh Prajna Bethany Mills 2 Institute for Regeneration and Repair, The University of Edinburgh , UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bethany Mills For correspondence: lalitha{at}aravind.org beth.mills{at}ed.ac.uk Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Aim To measure aetiology and antibiotic resistance (AMR) trends of bacteria cultured from corneal scrapings from patients with infectious keratitis at a tertiary referral hospital in South India. Methods In this retrospective study, bacterial aetiology and antimicrobial resistance profiles were identified from the microbiology records of patients undergoing microbial keratitis diagnosis at the Aravind Eye Hospital, Madurai, India from 2013-2024. Statistical analyses were performed by Spearman’s rank correlation coefficient to identify significant trends. Results P. aeruginosa (n=1047) was the most frequently isolated bacteria, followed by S. pneumoniae (n=987). There were significant increases in the number of P. aeruginosa (rs: 0.66; P=0.0219) and S. aureus isolates cultured (r s : 0.70; P=0.0130). S. aureus demonstrated increasing resistance to cefazolin (rs: 0.76; P=0.0015), gatifloxacin (rs: 0.75; P=0.0071), levofloxacin (r s : 0.60; P=0.0442), moxifloxacin (r s : 0.59; P=0.0437) and chloramphenicol (r s : 0.78; P=0.0049) over time. S. pneumoniae resistance towards tetracycline significantly increased (r s : 0.80; P=0.0029). P. aeruginosa isolates remained largely susceptible to all antibiotics screened, with significant decreasing resistance rates to ceftazidime (r s : −0.71; P=0.019), amikacin (r s : −0.59; P=0.0489), gentamicin (r s : −0.66; P=0.0219) and tobramycin (r s : −0.69; P=0.017) identified. No significant trends in resistance patterns were identified for Nocardia spp .. Conclusion Bacterial aetiology and antibiotic resistance rates shifted over time for key pathogens causing keratitis. Understanding these in the local context is important. For instance, while others in India have reported increasing P. aeruginosa AMR, we did not find this to be true in our patient population. This may have implications for local prescribing guidelines. What is already known on this topic Microbial keratitis remains a highly prevalent, sight-limiting condition in India. Treatment options are limited, and are confounded by emergent antibiotic resistance (AMR) of causative pathogens. What this study adds This study demonstrates shifting bacterial aetiology, and antimicrobial resistance patterns. Our data on P. aeruginosa resistance patterns contradict other reporting within India, where our resistance rates remained low. How this study might affect research, practice or policy Fluoroquinolones are the most common first-line treatment for bacterial keratitis, yet over 80% of our S. aureus isolates demonstrated resistance towards fluoroquinolones in 2024. Understanding local and temporal bacterial aetiology and resistance rates are imperative for designing local guidelines for patient treatment practices. Introduction Microbial keratitis is an ocular emergency and sight threatening condition affecting over 2 million people each year ( 1 , 2 ). Microbial keratitis may be caused by a wide variety of opportunistic pathogens; over 680 species of bacteria, fungi, virus and amoeba have been attributed to the disease ( 3 , 4 ). While this includes 250 bacterial species, the majority of cases are caused by a few species of gram-positive bacteria, primarily Staphylococcus aureus, Coagulase Negative Staphylococcus (CoNS) and Streptococcus pneumoniae ; and gram-negative Pseudomonas aeruginosa ( 5 , 6 ). Nocardia spp. is also reported as an emerging and important keratitis causing pathogen in some regions of India, where it has been reported as the third most prevalent bacterial species ( 7 , 8 ). Broad-spectrum, topical antibiotic therapy remains the gold-standard treatment approach for bacterial keratitis, often in the absence of a microbiological diagnosis. Worldwide, the most common treatment regimens utilise a fluoroquinolone as a monotherapy, or combination dual-therapy of cephalosporin, aminoglycoside and/or fluoroquinolone, as per local guidelines ( 6 , 9 , 10 ). However, antimicrobial resistance (AMR) of bacterial ocular isolates has become an increasingly concerning public health threat, complicating the treatment of microbial keratitis ( 5 , 6 , 11 ). Correlation between increased in vitro antimicrobial minimum inhibitory concentration (MIC) and worse clinical outcomes has been reported ( 12 - 15 ). The incidence of microbial keratitis, pathogen aetiology and AMR profile are each subject to geographic and temporal variations. It is critical to understand these trends globally and in the local context to inform policy and treatment guidelines. As recently reported by Khor et al ( 16 ), MIC analysis of Pseudomonas aeruginosa isolates obtained during their Asia Cornea Society Infectious Keratitis Study (ACSIKS) ( 17 ) demonstrated that isolates obtained from India were more likely to exhibit AMR compared to samples obtained elsewhere in Asia. However, their analysis combined data from across India, irrespective of geographic location. This is despite previous data from South India (collected between 2002-2012) indicating differential resistance patterns of bacterial isolates compared to samples obtained more recently in North, Central and Eastern India ( 18 - 22 ). Herein, we report contemporary aetiology of bacterial keratitis isolates, cultured from patients at a tertiary care centre in South India over a 12-year period (between 2013-2024), as well as report the trends in in vitro antimicrobial resistance and susceptibility during this time. Materials and Methodology We conducted a retrospective study, utilising microbiological records of patients diagnosed with microbial keratitis at a tertiary eye care referral centre in South India between January 2013 - December 2024. Microbial aetiology and antimicrobial resistance profiles of culture positive cases were extracted over the 12-year period. Only culture-positive bacterial keratitis cases were included; fungal and mixed infections were excluded. This study was approved by the Aravind Eye Hospital Institutional Review Board, Madurai. Microbiological identification of pathogens Corneal scraping specimens were cultured as per routine clinical practice on solid agar. Following growth, pure cultures of the organisms were used for species level identification through standard microscopic and biochemical tests. Further details are provided in the online supplementary methodology. Antibacterial susceptibility testing Each pure bacterial isolate underwent in vitro antibacterial susceptibility testing using the Kirby-Bauer disc diffusion method as per the CLSI guidelines ( 23 , 24 ). Details of antibacterial agents used are provided in the online supplementary methodology. Statistical analysis Spearman’s rank correlation coefficient (R s ) was used to measure the strength and direction of association between time and number of species identified, or % of isolates with resistance to each antibiotic. R s : ±0.00-0.19 = very weak correlation; ±0.20-0.39 = weak correlation; ±0.40-0.69 = moderate correlation; ±0.70-0.89 = strong correlation; and ±0.90-1.0 = very strong correlation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.0001; and **** P ≤ 0.00001. All analyses were performed using GraphPad Prism version 10 (GraphPad Software, USA). Results 1. Trends in bacterial isolation rate and aetiology A total of 22,887 patient corneal samples were cultured over the 12-year period, with a 53.3% culture positivity rate. 3728 (16.3%) had bacterial growth, 7891 (34.7%) had fungal growth, 189 (0.8%) had parasite growth, 358 (1.6%) had both bacterial and fungal growth, and 10,679 (46.7%) had no growth. In total, 4250 bacteria were isolated from patient corneal scrapes between 2013 and 2024. Bacterial culture rates remained stable over the study period (supplementary table 1, figure 1a , b). However, the number (P=0.0187), and therefore the overall proportion (P=0.0240) of gram-negative bacteria significantly increased over time from just 28% of cases in 2013, to 41% of isolates in 2024. Notably, there was a reduction in total isolates obtained in 2020, reflective of fewer patients attending the cornea clinic due to the COVID-19 pandemic. Download figure Open in new tab Figure 1. Bacterial isolates cultured from corneal scrape samples 2013-2024. (a) absolute number of isolates; (b) proportion of gram-positive and gram-negative isolates. (c) Absolute number and (d) proportion of gram-positive bacteria cultured from microbial keratitis patients. (e) Absolute number and (d) proportion of gram-negative bacteria cultured from microbial keratitis patients. Plots display bacteria with a minimum of 20 isolates cultured during the study period. Vertical dashed lines in (a, c and e) indicate where fewer samples were collected due to COVID-19 pandemic reducing patient numbers. Trends analysed by Spearman’s rank correlation coefficient. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. 1.1. Key gram-positive bacteria trends A total of 2627 gram-positive bacteria were cultured from the patient corneal scrapes (supplementary table S2 and S3). Streptococcus pneumoniae was consistently isolated with the highest frequency ( figure 1c ), contributing to an average of 38% of gram-positive cases ( figure 1d ). The frequency that S. aureus was cultured from patient cornea scrapes significantly increased over time (P=0.013), doubling from 8% in 2013 to 16% in 2024 (P=0.0005). Conversely, coagulase negative Staphylococcus (CoNS) trended downwards, from around 12% of cases between 2013-2015 to 6% in 2023 and 2024. Notably, Actinomyces spp. (P=0.004) and Propionibacterium acnes (P=0.0032) significantly increased in number and as a proportion of all gram-positive bacteria isolated. While only 23 Actinomyces spp . and 20 P. acnes isolates were cultured in total, 18 and 19 of these were cultured between 2020-2024, respectively (supplementary table S2 and S3). 1.2. Key gram-negative bacteria trends A total of 1623 gram-negative bacteria were isolated from the patient corneal scrapes (supplementary table S4 and S5). Pseudomonas aeruginosa was consistently isolated with the highest frequency ( figure 1e ). While cases of microbial keratitis caused by P. aeruginosa increased significantly over time (P=0.0219), this was proportional to the overall increase in gram-negative organisms cultured from patients, and P. aeruginosa contributed steadily to an average of 65% of gram-negative cases throughout the study period ( figure 1f ). Conversely, Enterobacteriaceae spp. numbers remained relatively stable over the course of the study, and therefore represented a decreasing trend in their proportion of gram-negative bacteria (P=0.0008), falling from 20% to 9% of gram-negative bacteria between 2013 and 2024. Both Aeromonas hydrophila (P=0.0003) and Haemophilus spp. (P=0.0037) were only cultured from patients from 2020 onwards, contributing to 42 and 20 cases respectively (supplementary table S4 and S5). 2. Trends in antibiotic resistant rate 2.1. S. aureus 290 S. aureus isolates were evaluated for methicillin sensitivity (via assay with cefoxitin) ( 25 )), and 246 (84.8%) of the isolates underwent further susceptibility testing. In total twelve antibiotics were screened, from six antibiotic classes ( figure 2a , supplementary tables S6 and S7). Increasing antimicrobial resistance patterns were identified for a number of the antimicrobial classes evaluated over the study period. Download figure Open in new tab Figure 2. Antimicrobial resistance rates of bacterial isolates cultured from corneal scrape samples 2013-2024. Collected isolates underwent antimicrobial sensitivity testing. Data are presented as the % of all (a) S. aureus ; (b) S. pneumoniae ; (c) Nocardia spp; and (d) P. aeruginosa isolates with resistance to antibiotics. Resistance determined by CLSI breakpoints (or those as outlined in the supplemental methodology section). Trends analysed by Spearman’s rank correlation coefficient. * P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. The number of isolates evaluated per bacterial species are reported in supplementary table S6. We found that while the number of methicillin resistant (MRSA) S. aureus isolates did significantly increase (P=0.0095) over the study period, this was not significant as a proportion of all S. aureus isolates due to concurrent increasing incidence of MSSA (supplementary tables S2 and S3). High rates of resistance towards fluoroquinolones were reported throughout our study, which steadily increased over time from ∼60% of isolates in 2013, to ∼80% of isolates in 2024 (reaching significance for gatifloxacin (P=0.0071), levofloxacin (P=0.0442), and moxifloxacin (P=0.0473)). While sensitivity rates towards chloramphenicol remained above 94% until 2021 (with two spikes in resistance in 2017 and 2019), the number of resistant isolates subsequently increased significantly, with one third of isolates resistant to chloramphenicol in 2024 (P=0.0049). Resistant rates were low in 2013 to gentamicin and tobramycin, subsequently increasing rapidly and remaining relatively stable (with a reduction in 2017 and 2018) over the next decade. An average 25% of isolates were resistant to gentamicin and 30% of isolates resistant to tobramycin between 2019 and 2024. None of the 246 clinical S. aureus isolates evaluated demonstrated resistance to vancomycin. Of note, during the study period, local guidelines for interpreting the cefazolin zone of inhibition were updated. Prior to the change in breakpoint (2022), no incidences of resistance were recorded. However, after the reclassification, increasing year-on-year resistance has been reported, from 6% to 55% of isolates resistant to cefazolin. 2.2. S. pneumoniae Antibiotic resistance data were available for 974 (98.6%) of the S. pneumoniae clinical isolates. The isolates were screened against ten antibiotics from five classes ( figure 2b , supplementary tables S6 and S8). Resistance towards tetracycline was the most commonly found resistance for S. pneumoniae in 2013, observed in 32% of isolates. Over the study period, resistance rates continued to trend upwards, with a peak in resistant isolates reaching 59% in 2022 (P= 0.0029). This was the only antimicrobial for which there was a significant trend. While resistance towards the fluoroquinolones was less frequently observed, there was a peak in resistance towards gatifloxacin and ofloxacin 2017-2019, where over one third of isolates were resistant. This peak was followed by a sharp decrease in resistant isolates, with fluoroquinolone resistance observed in around 10% or fewer isolates across 2020-2024. S. pneumoniae remained highly sensitive to cefotaxime throughout the study period, with 99% of isolates showing sensitivity. Similarly, resistance towards chloramphenicol remained low and stable, with an average of 96% of isolates showing sensitivity. None of the isolates demonstrated resistance to vancomycin. 2.3. Nocardia spp Antibiotic resistance data were available for 270 (97.8%) of the Nocardia spp. isolates. Twelve antibiotics from five classes were evaluated ( figure 2c , supplementary tables S6 and S9). High levels of resistance prevailed for the Nocardia isolates. Notably, the Nocardia isolates showed a largely erratic resistance pattern to the majority of antimicrobials tested, and there were no significant changes over time. Overall, aminoglycosides remained the most effective class of antibiotics against Nocardia , with ∼75% of isolates sensitive to gentamicin and tobramycin, and 98% to amikacin. An average of 70% of Nocardia spp. isolates were resistant to vancomycin, the rate of resistance was relatively stable between 2013-2021 (except for 2018), however the rate of resistance decreased dramatically over the last three years; an average of 80% of isolates showed resistance 2013-2021 (excluding 2018), dropping to an average of 55% isolates resistant from 2022-2024. 2.4. P. aeruginosa Antibiotic resistance data were available for 1045 (99.8%) of the P. aeruginosa isolates. The isolates were evaluated against eleven antibiotics, from five classes ( figure 2d , supplementary tables S6 and S10). Overall, P. aeruginosa remained sensitive to the majority of antibiotics tested, with >90% of isolates sensitive to aminoglycosides (amikacin, gentamicin and tobramycin); fluoroquinolones (ciprofloxacin, levofloxacin, and moxifloxacin); and polymyxin B. Excluding the aminoglycosides, for each of these antibiotic classes, rates of resistance remained mostly stable across the study period. However, there was a larger peak in resistance in 2016 towards aminoglycosides (around 25% of isolates showed resistance), followed by a significant decline in resistant rates, to less than 5% in from 2021 (amikacin: P=0.0489; gentamicin: P=0.0219; and tobramycin P=0.017). Notably, significantly fewer isolates showed resistance towards ceftazidime over the study period, from 8% in 2013, to 3% in 2024 (P=0.0129). Discussion Microbial keratitis prevalence and treatment remains a significant challenge in South India. While we determined that the five most frequently cultured bacteria were P. aeruginosa (25%); S. pneumoniae (23%); Corynebacterium spp. (9%); S. aureus (7%) and Nocardia spp. (6%) from our South Indian cohort, a similarly sized study from North India found coagulase-negative S. aureus to be the most prevalent organism (61%), followed by P. aeruginosa (14%), S. aureus (14%), Enterobacteriaceae (9%) and Streptococcus spp. (2%) ( 20 ). This differential aetiology was also observed in Central India, where S. pneumoniae was the most prevalent bacteria (43%), followed by S. aureus (20%), Bacillus spp. (15%) and P. aeruginosa (12%) ( 26 ). Notably, a 30 year study from another large centre in South India found that their S. pneumoniae cases increased over time, and was the only bacteria to show a significant trend between 1991 and 2020 ( 27 ), in contrast, our hospital did not see a significant increase in S. pneumoniae culture rates over a 22 year period (between 2002-2012 ( 18 ), or in our current study between 2013-2024). In addition to differential aetiology, varying antibiotic susceptibility profiles were reported across each of these Indian regions. This further underscores the need to understand the specific geographical, local and temporal context for microbial keratitis diagnosis and treatment strategy. Overall, our study identified shifting trends in antimicrobial resistance rates for several antibiotics of clinical relevance, for some of the most prevalent bacterial species causing microbial keratitis. Particularly, increasing trends were identified for S. aureus across multiple drug classes, and somewhat unexpectedly, decreasing trends in resistance were identified for P. aeruginosa isolates. As has been identified by us and others ( 21 , 28 , 29 ), there was a high level of S. aureus resistance towards fluoroquinolones, even at the outset of this study. We found that the resistance rates continued to increase over time, with over 80% of isolates resistant to each of the fluoroquinolones tested (levofloxacin, gatifloxacin, moxifloxacin and ciprofloxacin) in 2024. It has been reported that S. aureus fluoroquinolone resistance is correlated with increasing incidence of multi-drug resistant MRSA strains ( 28 , 30 ). However, our relative rates of MRSA remained stable throughout the study period, indicating that increased fluoroquinolone resistance was also attributable to MSSA strains, although these data were not delineated in our study. Increasing fluoroquinolone resistance is particularly concerning for the management of patients, with 0.5% moxifloxacin monotherapy being the first-line treatment for bacterial corneal ulcers (excluding Nocardia ). Moreover, it has previously been reported that clinical isolates from patients who had been treated with fluoroquinolones prior to having their sample collected exhibited higher MICs, compared to clinical isolates obtained from treatment naïve patients ( 31 ), indicating that increasing resistance could be driven by the therapy itself. Furthermore, higher in vitro moxifloxacin MIC values have been associated with worse clinical outcomes for bacterial keratitis patients ( 12 ). While caution should be exercised comparing S. aureus resistance patterns towards cefazolin pre and post 2022 due to changing breakpoint guidance, it is notable that the number of isolates with resistance has steadily trended upwards year-on-year since 2022, with over half of isolates showing resistance in 2024. Fortified cefazolin remains a first-line treatment for bacterial keratitis and therefore this emerging resistance should be monitored closely. Although we did see an increase in the absolute numbers of MRSA isolates, the number of MSSA also increased. Reassuringly, we did not see an increase in the proportion of MRSA over the study period (after a significant increase between 2002-2012 ( 18 )). Between 2013-2024, 27% of isolates demonstrated methicillin resistance, which is within the range of other studies from Asia ( 28 , 32 ), and the USA ( 30 ), who have also reported a stabilisation of MRSA isolates. S. pneumoniae and P. aeruginosa were the most frequently cultured bacteria within our study; accounting for almost half of bacterial keratitis isolates (23% and 25% respectively). In contrast to S. aureus and Nocardia spp., these isolates exhibited higher levels of in vitro sensitivity, and temporal stability to most of the antibiotics tested. Notably, while others have reported increasing antibiotic resistant P. aeruginosa from microbial keratitis patients in India in recent years, particularly in northern and central regions ( 16 , 20 - 22 ), we did not find this trend to be true in our South Indian population. On the contrary, there was on average, 90% susceptibility to each antibiotic, and surprisingly resistance rates towards ceftazidime, amikacin, gentamicin and tobramycin significantly declined over the last 12 years. While others in more northerly regions of South India also reported 30% resistance rates of P. aeruginosa isolates to ceftazidime ( 27 ), curiously, our observations reflect those found in other regions across Asia ( 16 , 32 ), and further underscore the need for assessing AMR in the local context. Our study has many strengths, including the large sample size, 12-year study period and contemporary data reporting. In a landscape of rapidly fluctuating antimicrobial resistance, swift time-to-reporting is essential to identify emerging pathogens and resistance profiles, with which to closely monitor and guide treatment decisions, for example, our recently observed S. aureus resistance towards cefazolin, and S. pneumoniae resistance towards tetracycline. However, an inherent limitation in interpreting antimicrobial resistance data in a clinically meaningful way is the paucity of MIC breakpoint guidelines in the context of topical, frequently applied eye-drops, where local concentration is likely to be higher than for antibiotics delivered systemically. Thus, we (and others) may have over reported clinically relevant antibiotic resistant rates. Nonetheless, the breakpoints that we have utilised are common with other studies reporting on microbial keratitis isolates. Despite this, work to determine clinically relevant breakpoints are warranted to facilitate rational clinical guidelines. Data Availability Data are available on reasonable request to the corresponding authors. Acknowledgements BM and LF were supported by a UKRI Future Leaders Fellowship (No. MR/V026097/1). Footnotes Synopsis/Precis: We report aetiology and antimicrobial susceptibility patterns of bacteria cultured from microbial keratitis patients in South India between 2013-2024. Isolate frequency and antimicrobial resistance patterns have changed significantly for several common bacteria. Data availability statement: Data are available on reasonable request. Competing Interests: none. References 1. ↵ Hossain P. Microbial keratitis-the true costs of a silent pandemic? Eye (Lond) . 2021 ; 35 ( 8 ): 2071 – 2 . OpenUrl PubMed 2. ↵ Brown L , Leck AK , Gichangi M , Burton MJ , Denning DW . The global incidence and diagnosis of fungal keratitis . The Lancet Infectious Diseases . 2021 ; 21 ( 3 ): e49 – e57 . OpenUrl CrossRef PubMed 3. ↵ Bartimote C , Foster J , Watson S. The spectrum of microbial keratitis: an updated review . The Open Ophthalmology Journal . 2019 ; 13 ( 1 ). 4. ↵ Ung L , Bispo PJ , Shanbhag SS , Gilmore MS , Chodosh J. 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