Antibacterial Susceptibility Pattern of S. maltophilia Isolates at A Tertiary Care Hospital, India

A sudden emergence of Stenotrophomonas maltophilia as a primary pathogen both in immunocompromised and immunocompetent individuals has raised a serious concern, as it is associated with significant case fatality ratio. We intended to study the clinico-microbiological profile of S. maltophilia isolates from various samples and outcome of the infections in a tertiary healthcare center, Pune, India. This is an observational cross-sectional study was conducted from January 2021 to June 2022 at Department of Microbiology of a tertiary care Centre in Pune, India. Of the 12049 samples received for culture, S. maltophilia was isolated in 57 samples. Only 42 samples with pure growth of S. maltophilia were included in the study with 15 excluded due to mixed growth. All isolates were confirmed by VITEK-MS (bioMerieux, SA, France) which uses Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology. Of the 42 isolates, majority were isolated from pus(28.6%) and most of patients (61.9%) were from acute health care settings. The isolates had high susceptibility to Cotrimoxazole (85.7%) and Minocycline (85.7%) and low susceptibility to Ceftazidime (45.2%). A case fatality rate of 7.1% (3/42 cases) was noted and 39 cases were discharged after complete treatment. All the three fatal cases were susceptible to levofloxacin, ciprofloxacin, cotrimoxazole and minocycline and all three fatal cases were resistant to ceftazidime. S. maltophilia has recently shown an increase in nosocomial infections especially in acute healthcare settings like ICU and other critical care wards. The isolates of the present study had high susceptibility to trimethoprim-sulfamethoxazole (TMP-SXT) and Minocycline and low susceptible to Ceftazidime.


INTRODUCTION
S. maltophiliais an aerobic, gramnegative bacterium, traditionally considered as an environmental contaminant (increasingly isolated in hospital settings from different sources like haemodialysis water and dialysate samples,contact lens solutions, sink drains, faucets,hand-washing soap , contaminated chlorhexidine-cetrimide topical antiseptic and tap water) and is also known to produce biofilms. 1,2.maltophilia is a nosocomial pathogen especially in immunocompromised but community acquired S. maltophilia infections in comorbid patients is also reported worldwide.3 A sudden emergence of S. maltophilia as a primary pathogen both in immunocompromised and immunocompetent individuals has raised a serious concern, as it is associated with significant case fatality ratio.4 S. maltophilia is commonly associated with bacteraemia, endocarditis, respiratory tract infections like pneumonia, mastoiditis, peritonitis and exit sign infections in patients undergoing peritoneal dialysis, soft tissue and skin infections, joint infections and Central Nervous System (CNS) infections.5,6 At risk population are patients with immunocompromised states like diabetes mellitus, cancer patients on treatment, patients with indwelling devices.S. maltophiliais an emerging pathogen in patients with cystic fibrosis which unlike Pseudomonas, Burkholderia spp is multidrug resistant.7 This organism has ability to form biofilms on moist surfaces such as respiratory tubing,hospital water plumbing systems, dental suction tubing, Intra Venous (I.V) lines, dialysis equipment,catheters,domestic sink drains,clinical sink drains, and faucets.8 The transmission of S. maltophilia to susceptible individuals may occur through direct contact with the source. 9S. maltophilia can be identified by automated methods like VITEK.Lack of automated systems hampers the early diagnosis leading to prolongation of illness and Intensive Care Unit(ICU) stay. 10 Studies from India have shown that 90.9% S. maltophilia were susceptible to colistin and 27.3% susceptible to ceftazidime and minocycline.11 S. maltophilia is primarily isolated in patients in the intensive care unit and need for vasopressors, autoimmune disease, lower P/F ratios and thrombocytopenia were associated with higher mortality.12 We intended to study theclinicmicrobiological profile of S. maltophilia isolates from various samples and outcome of the infections in our tertiary healthcare center.

METHODOLOGY
This is an observational and crosssectional study which was carried out at Department of Microbiology of a tertiary care centre in Pune, India between 01 Jan 2021 till 30 Jun 2022.Ethical committee approval was obtained from the institution.All cases found positive for S. maltophilia were included in the study and samples with mixed type of growth were excluded.Sample collection was done as per standard protocol and was transported immediately to the laboratory.Sample collection was done only once per patient.
All samples (except blood and urine) were inoculated on Blood agar and MacConkey agar where as urine samples were inoculated on Cysteine Lactose Electrolyte Deficient agar and blood was inoculated in BACT/Alert blood culture bottles and all the samples were incubated at 35-37°C.Isolates were identified using Matrix assisted Laser Desorption/ionization-time of fight(MALDI-TOF), Mass spectrometry (MS) or VITEK 2 Compact.Antimicrobial susceptibility testing was performed as per Clinical Laboratory Standards Institute (CLSI) guidelines M100 31 st and 32 nd edition.

MALDI-TOF MS identification
Strains were identified by MALDI-TOF MS principle using the VITEK MS (bioM‫י‬rieux SA, France).Isolates were smeared onto the sample spots on the target slide and 1µL VITEK MS-CHCA matrix was applied over the sample and air dried till both the matrix and sample co-crystallized.The target slide was loaded into the VITEK MS system to acquire the mass spectra and data was compared with known mass spectra contained in the database.

Staphylococcus aureus ATCC 25923 and
Escherichia coli ATCC 25922 were used as the quality control strain.

VITEK 2 susceptibility testing
The isolates were subjected to Antimicrobial susceptibility testing using VITEK 2 (bioMerieux SA, France).The cards used were GN AST 280 and 281.Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the quality control strain.Quality controls of all the bacteriological AST procedures are carried out on a weekly cycle.

Statistical analysis
Data was tabulated and analyzed using software Open Epi version 3.01 and Statistical Package for Social Sciences (SPSS) version 22. Categorical data have been presented as numbers and percentages (%) and quantitative data in terms of mean and standard deviation.Categorical variables have been analysed using Pearson's chi-square test and Fisher exact tests (when the expected count of 20% of cells is less than 5).A p value of <0.05 has been considered as statistically significant.

RESULTS
Forty-two culture positive cases were identified and included in the study.Table 1 shows the list of various clinical specimens from where S. maltophilia was isolated.
The mean age of study subjects was 48.6 ± 12.1 years with a third of patients in the age group of 41 to 50 years (14 cases, 33.3%), followed by 31 to 40 years (12 cases, 28.6%).There was a male predominance with a ratio of 6:1(36 men and 06 women).
Majority of isolates (61.9%) were from patients admitted in Acute healthcare settings including Intensive Care Units (ICU), High Dependency Units (HDU)and acute wards.Amongst the non-ICU patients S. maltophilia was isolated from surgical wards and Cancer wards.Sixteen cases (38.1%) had an immunocompromised s ta t e , w h i l e re m a i n i n g ( 6 1 .9 % ) w e re non-immunocompromised.
The isolates had high susceptibility to trimethoprim-sulfamethoxazole (TMP-SXT) (85.7%) and Minocycline (85.7%) and least susceptible to Ceftazidime (45.2%).The isolates found to be susceptible to all drugs were 33.33% while 16.7% of isolates were resistant to Ciprofloxacin, Minocycline and Ceftazidime.The drug susceptibility among fluoroquinolones was comparable with levofloxacin having slightly higher (76.9%) sensitivity than ciprofloxacin (71.4%).Amongst isolates 19.04% were resistant to both Fluoroquinolones (Levofloxacin, ciprofloxacin) tested.Antibiogram of isolates is depicted in Table 2.
A case fatality rate of 7.1% (3/42 cases) was noted in the present study among patients  with S. maltophilia and the remaining 39 cases were discharged after complete treatment.We further tried to explore the association of factors that may be responsible for mortality among the cases.Case fatality in the age group of 41 to 50 years and 51 to 60 years was observed to be 14.3% and 11.1%, respectively, with no deaths in the age group of 31 to 40 years and more than 60 years.All three deaths that occurred were among men.In the present study, the case fatality rate (CFR) was significantly higher among cases with one or more comorbidities (33.3%), while the CFR among patients without co-morbidity was 2.8%.Similarly, CFR among patients with multi-system involvement was 25% and all cases with single organ involvement were discharged after complete treatment (Table 3).On comparison of final outcome (death or discharge) with drug susceptibility, it was noted that among the three cases with ill-fated outcome all were sensitive to levofloxacin, ciprofloxacin, cotrimoxazole and minocycline.Drug resistance among survival cases varied from 15.4% to 30.8% for these drugs with least drug resistance being in cotrimoxazole, minocycline (15.4%) and ciprofloxacin having a drug resistance of 30.8%.On the contrary to the above findings, all three fatal cases were resistant to ceftazidime (100%), and among discharged cases, 51.3% of cases were resistant to the drug (Table 4).
One interesting finding in our study was isolation of S. maltophilia from bile in a 72 year old male patient of cholelithiasis, patient had no comorbidities.The isolate was sensitive to all the drugs tested.Patient was treated with Co-trimoxazole (TMP-SMX), later operated, and discharged.
Resistance to the drug ceftazidime was seen in all the three cases succumbing to death.Though the cause of death was multifactorial, and all three patients had comorbidities, the slightest delay in the initiation of appropriate antibiotic was the major contributor to the ill-fated outcome.
The resistance patterns of S. maltophilia was comparable with studies done by Hafiz TA  et al., where 95.9% of samples were susceptible to Trimethoprim-Sulfamethoxazole, followed by 68.9% of samples to levofloxacin.The least susceptibility was seen with ceftazidime (33.1%). 14ew treatment guidelines recommend combination of antibiotics to obtain synergic effect. 15Few studies have shown the synergic effect with Trimethoprim-Sulfamethoxazole and Tigecycline, and between Amikacin and Tigecycline. 16It is also observed that when TMP-SMX is combined with either ceftazidime, ciprofloxacin or tobramycin, it produces higher bactericidal efficacy against S. maltophilia clinical isolates. 17Moxifloxacin has shown some promising effects for the treatment of multi drug resistant S. maltophilia infections in some of the studies. 18 n o n e o f t h e r e c e n t s t u d i e s , S. maltophilia isolates were resistant to at least six of the antibiotics tested, including Trimethoprim/ Sulfamethoxazole (SXT). 19However, Tigecycline which has good in vitro activity against trimethoprim/sulfamethoxazole resistant strains, is a promising alternative for treating S. maltophilia infections. 20

Limitations of the study
The present study is single centric study with smaller sample size of only 42 isolates.Moreover, only 3 isolates were ceftazidime resistant, and disease outcome prediction cannot be concluded on such a small sample size.None of the samples were clones, as all three samples were from different patients and from different wards.However, the study provides an opportunity for further investigation on the role of ceftazidime resistance and mortality.

CONCLUSION
The mean age of present study subjects was 48.6 ± 12.1 years, with a third of patients in the age group of 41 to 50 years and male predominance with a ratio of 6:1 was observed.Majority of isolates were from patients admitted in Acute healthcare settings like Intensive Care Units (ICU), High Dependency Units (HDU) and Namaji et al | J Pure Appl Microbiol.2023;17(4):2256-2262. https://doi.org/10.22207/JPAM.17.4.23

Table 1 .
Demographic details and sample report of patients *Statistically significant

Table 4 .
Comparison of drug susceptibility with final outcome among study subjects