Original article / research
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Incidence Rate and Antibiotic Susceptibility Pattern of Listeria Species in High Risk Groups |
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Geethavani Babu, Balamuruganvelu S, Saleel V Maulingkar, R Srikumar, Sreenivasalu Reddy V, Senthamizhan VS 1. Assistant Professor, Department of Microbiology, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India. 2. Professor, Department of Microbiology, DM Wayanad Institute of Medical Sciences, Wayanad, Kerala, India. 3. Assistant Lecturer, Department of Microbiology, Goa Medical College, Bambolim, Goa, India. 4. Research Associate, Department of Microbiology, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India. 5. Professor, Department of Microbiology, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India. 6. Student MBBS, Sri Lakshmi Narayana Institute of Medical Sciences, Puducherry, India. |
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Correspondence
Address : Dr. Geethavani Babu, No.10 9th Cross Extension, Rainbow Nagar, Puducherry-605011, India. E-mail: gee192@gmail.com |
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ABSTRACT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
: Listeriosis, a bacterial food borne disease caused by Listeria spp. leads to mild food poisoning in the healthy individuals and severe systemic disease in immuno-compromised patients, pregnant women and extremes of age. Listeriosis in India largely remains ignored, even though there is an increase in the prevalence worldwide. Aim: To determine the incidence rate of Listeria spp. in clinical samples collected from various immuno-compromised individuals and to study its antibiotic susceptibility pattern. Materials and Methods: Total 643 clinical samples from high-risk group individuals were tested for Listeria spp. using standard culture and identification methods. Results: The overall incidence rate of Listeriosis in high risk individuals was 4.98%. The incidence rate in pregnant women and women with bad obstetric history was found to be 11.04%. Listeria spp. was found in 10.2 % of gastroenteritis cases, which is one of the major concerns in high risk groups. Increased resistance to clindamycin 68.8%, followed by penicillin G 37.5%, erythromycin 31.3% and ampicillin 25 % was found. Conclusion: In view of the high incidence rate of Listeriosis in our setting, it should be considered as a differential diagnosis in the high risk groups and diagnostic capability for the pathogen needs to be strengthened. Furthermore, increased antibiotic resistance is a cause for concern and the trends need to be monitored. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords : Immuno-compromised patients, Listeria monocytogenes, Listeriosis, Pregnancy | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INTRODUCTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Listeriosis, a potentially serious invasive bacterial food borne disease caused by genus Listeria which leads to mild food poisoning in healthy individuals and severe systemic disease in certain well-defined high-risk groups.The genus Listeria includes multiple species namely L.monocytogenes, L.ivanovii, L.innocua, L.fleischmannii, L.welshmeri, L. seeligeri, L.grayi, L.marthii and L.rocourtiae (1),(2). But among different species only L.monocytogenes and L.ivanovii are pathogenic in humans (3). Listeria monocytogenes has been found to be the causative agent in several outbreaks of food-borne Listeriosis (4). L.ivanovii infection in humans is although rare, but there are reports on isolation of this organism from cases of AIDS and abortion (5). Immunocompromised individuals including transplant patients, dialysis patients, patient on immunosuppressive therapy, HIV patients (6), cancer patients (7), pregnant women (8), infants and neonates (9) are reported to be at high risk of getting Listerial infection. Listeriosis is a serious infection with high case fatality rate of about 20-30%, neonatal death rate 50% and hospitalization rate of about 91% (10). In view of the high prevalence of Listeria monocytogenes in foods, together with the high mortality rate, this pathogen represents an important human health hazard (11). Reports also suggest that incidence of Listeriosis has been increasing world-wide (12). Besides, Listeriosis in India largely remains ignored. The literature reviews pertaining to the Listerial infections among immuno-compromised high risk groups in the Indian subcontinent is scarce. L.monocytogenes infections are usually treated with a single antimicrobial agent and combined therapies are recommended for the treatment of immuno-compromised patients (13). Generally penicillin, ampicillin, amoxicillin, cotrimoxazole, tetracycline, chloramphenicol or aminoglycosides are recommended for the treatment of Listerial infection (14). In 1988, the Multidrug resistant L.monocytogenes was first reported in France (15). Since, then the number of drug resistant strains has been continually increasing (16). The present study was undertaken to determine the incidence rate of Listeriosis in certain high risk groups. Furthermore, we aimed to determine the drug susceptibility pattern of Listeria spp. towards the common antibiotics used in the treatment of Listeriosis. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MATERIAL AND METHODS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A cross-sectional study conducted over a period of 1 year from June 2014 to May 2015 in the Department of Microbiology, Sri Lakshmi Narayana Medical College, Hospital and Government General Hospital, Puducherry, India. This study was approved by the Institutional Human Ethics Committee. Informed consent was obtained from all participants included in the study. Patient population included HIV patients, patients with malignancies, chronic liver disease, chronic renal failure, patients on long term corticosteroid therapy, infants (<12 months of age), pregnant women, women with spontaneous abortions or stillbirths and elderly patients (>65 years) presenting with either fever, flu-like illness, signs and symptoms of meningitis or diarrhea. A total of 643 clinical samples comprising 345 - blood samples, 65 - CSF, 17 - other body fluids, 49- diarrheal stool, 138 - amniotic fluid, 14 - placental bit and 15 - abortus material were aseptically collected. All the collected clinical samples were immediately transported to the laboratory and were processed for the isolation of Listeria spp. following the US Department of Agriculture (USDA) method (17). The samples were enriched by two step enrichment procedure by inoculating in University of Vermont medium (UVM)-1 and incubated at 30°C for 24 hours followed by (UVM)-2 and incubation at 30°C for 24 hours. A loopful of inoculum from enriched (UVM)-2 was cultured on to selective medium PALCAM agar and then incubated at 37°C for 24 hours. Grayish glistening colonies surrounded with a diffuse black zone were identified as Listeria colonies (Table/Fig 1) (17),(18). The identified Listeria colonies were examined morphologically for Gram positive coccobacilli (Table/Fig 2) and its characteristic tumbling motility at 20-25°C, which was then confirmed for genus Listeria on the basis of Latex agglutination test using LK07- Hi Listeria Latex Test Kit {Hi-Media, India}. The identified Listeria isolates were further subjected to biochemical characterization using KB012A-Hi Listeria identification kit which includes catalase test, nitrate reduction test, esculin hydrolysis, Voges-Proskauer test, methyl red test and sugar fermentation tests. The kit contained sugars like xylose, lactose, glucose, alpha-methyl-D mannoside, rhamnose, sucrose and mannitol to differentiate various species of Listeria. Isolates exhibiting catalase, methyl red and Voges Proskauer test positive and nitrate negative reactions were considered as “presumptive” Listeria isolates. These “presumptive” Listeria isolates were further differentiated up to the species level into L. monocytogenes and other Listeria species based on sugar fermentation pattern. Isolates which showed glucose, a-methyl - D mannoside, rhamnose, lactose and sucrose positive, xylose and mannitol negative were considered as L.monocytogenes. The recovered isolates were subjected to antimicrobial susceptibility testing by Kirby Bauer disc diffusion assay, as per CLSI guidelines 2012, with the following antibiotics generally recommended for the treatment of the Listeria infection such as ampicillin (10 mcg), penicillin G (10 units), tetracycline (30 mcg), chloramphenicol (30 mcg), trimethoprim/sulfamethoxazole (co-trimoxazole) (25 mcg), gentamicin (10 mcg), amikacin (30 mcg), erythromycin (15 mcg) and clindamycin (2 mcg). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESULTS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Out of 643 samples tested for Listeriosis, 32 samples showed the presence of Listeria spp. giving an overall incidence rate of 4.98%. Invasive Listeriosis had an incidence rate of 4.2% compared to Non-Invasive Listeriosis which had an incidence rate of 0.8% (Table/Fig 3). Abortus material (13.3%) and amniotic fluid (11.6%) yielded the highest rate of isolation followed by diarrhoeal stool (10.2%) (Table/Fig 4). Of the high risk groups, included in this study the incidence rate was highest among pregnant women and women with bad obstetric history (11.04%) followed by the elderly (7.69%) and HIV patients (5.17%) (Table/Fig 5). Biochemical characterization revealed that (21.9%) of Listeria isolates were L. monocytogenes (Table/Fig 6). Highest overall resistance rates among the isolates were to clindamycin (68.8%), followed by penicillin G (37.5%) and erythromycin (31.3%) (Table/Fig 7). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DISCUSSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In the present study the overall incidence rate in high risk individuals was 4.98%, which is higher compared to previous studies, where incidence rates of only 0.1 % and 11.3/1,000,000 Listerial cases were reported (10),(19),(20). This may be because we also included Listeria species other than Listeria monocytogenes when calculating the incidence rates.The percentage of L. monocytogenes in our study was 21.9 %, compared to other Listeria spp. which accounted for 78.1%. Further, pathogenicity studies are essential to prove the pathogenic potential of other Listeria spp. isolated in this study. Listeriosis was found to be high among pregnant women, older adults and HIV patients. These results are in accordance with the other published reports who reported an increasing incidence of Listeriosis in these groups, especially in elderly population and pregnant women (9),(21),(22),(23),(24),(25),(26),(27). In our study, Listeria spp. was isolated from 13.3 % of the abortus specimens. This is in accordance with other studies from India which reported a positivity rate of 1.34% - 4% from pregnant women and 14% from women with bad obstetric history (20),(28),(29). Maternal Listeriosis is usually mild but it is highly severe and fatal for the neonates (22),(30). Hence, the present study emphasizes the need to screen for Listeriosis in all stages of pregnancy. We found Listeria spp. in 10.2% of gastroenteritis cases among high risk groups, whereas another study has reported 20 –25% incidence of Listeria spp. in gastroenteritis cases among high risk groups (31). Listerial gastroenteritis is usually self -limiting in healthy individuals, but it is of major concern in case of immunocompromised individuals. Incidence of antibiotic resistance was currently low in L.monocytogenes compared to other Listeria spp. in our study. This study also observed relatively low percentage of tetracycline resistance in L. monocytogenes (14.3%), the most widely reported resistance (14),(32),(33). Overall increased resistance among Listeria genus to clindamycin (68.8%), penicillin G (37.5%), erythromycin (31.3%) and ampicillin (25%) was found. These resistance rates have to be interpreted with caution owing to the less number of isolates. Ampicillin resistance in clinical Listeria has been previously proved due to transfer of plasmids. Moreover increased clindamycin ampicillin and penicillin resistance has also been reported from food and other environmental sources (14),(15),(32),(33),(34), so it is possible that Listeria could have acquired drug resistance genes from multiple sources. Limitations All Listeria spp. isolated in the study were taken in to account. Biochemical speciation of non-Listeria monocytogenes isolated in this study was not done. Further, pathogenicity studies are required to prove the importance of non - Listeria monocytogenes isolated in this study. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CONCLUSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In view of the increased incidence of Listeriosis, this disease should be considered an important differential diagnosis in clinical practice especially in high risk individuals. Maternal Listeriosis should be considered in all stages of pregnancy and due diligence must be followed in laboratory diagnosis. Being a food borne pathogen, strong efforts have to be made to ensure food safety. The increased drug resistance in this genus to the commonly prescribed antibiotics against Listeriosis is an area of concern and judicious use of antibiotics is to be encouraged to prevent further increase in resistance. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACKNOWLEDGEMENT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
This work was supported by the grants received from ICMR, India (Project No : 2015 -04297) and SLIMS, Puducherry. The authors are thankful to Dr.K.V.Raman, Director (Health), Government of Puducherry, Dr. S. Basalingappa, Associate Dean, SLIMS Puducherry and Dr. R. Chidambaram, Director R&D Medical, SLIMS, Puducherry for providing the facilities and necessary support to carry out the work. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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