Molecular Characterization of Citrobacter freundii Isolated from Neonates in Neonatal Intensive Care Unit of Nepal

Introduction: Nosocomial Citrobacter spp. is emerging as a successful nosocomial pathogen in neonates in Nepal. The important risk factor being poor infection prevention and control practices. The objective of this study was to investigate the clonal relatedness of Citrobacter freundii isolated from clinical and nonclinical sources in Neonatal Intensive Care Unit (NICU) and to determine the presence of Extended Spectrum Beta-Lactamase (ESBL) genes and class 1 integron element. Materials and Methods: Polymerase chain Reaction (PCR) and PCR-Randomly Amplified Polymorphic DNA typing of the isolates were performed in three isolates to amplify class 1 integron element integrase gene, ESBL genes, and to study the clonal relatedness, respectively. Results: Two isolates harbored class 1 integron element. The blaCTX-M was present in all isolates and blaTEM-1 was present in one isolate. An isolate carried blaCTX-M and blaTEM-1 genes. All of these isolates were not clonally related. Conclusion: The study for the first time documented the emergence and spread of ESBL genes and class 1 integron element in multidrug resistant C. freundii in Nepal and urge for monitoring and surveillance of these strains.


Introduction
A mong nosocomial pathogens causing nosocomial infections, Staphylococcus aureus, coagulase negative Staphylococcus spp., Acinetobacter spp., Pseudomonas spp., Escherichia coli, Kleibsella spp., Enterobacter spp., and Citrobacter spp.are most common 1 .Citrobacter nosocomial infection in neonates is a cause of substantial rise in morbidity and mortality of neonates in the hospital in Nepal and globally 1,2,3,4 .The poor infection prevention and control practices have been a major determining factor for the spread of this nosocomial pathogen 1 .The infection in neonates is either horizontally transferred as a nosocomial infection or vertically transferred from mother during delivery 5 .The imprudent and less potent use of antibiotics has also led to the emergence of the multidrug resistant nosocomial pathogens 6 .Numerous studies have already determined the possession of class 1 integron element and ESBL genes in these isolates.

Material and Methods
The study had isolated 12 multidrug resistant Citrobacter spp.from various clinical and non-clinical sources.They were grouped into fi ve antibiotypes (I to V).The isolate C109, isolated from the nasal prong and C316709 were grouped with four other isolates in antibiotype I.These isolates along with C3409 belonging to antibiotype V were selected for the present study.The identifi cation of C. freundii was carried out as described previously 15 .Hydrogen sulfi de production on Triple Sugar Iron Agar slant along with other biochemical tests was used to confi rm as C. freundii.Overnight grown bacterial colonies were emulsifi ed in 1 ml sterile distilled water and boiled for 1 minute.The emulsifi ed colonies were centrifuged at 13,000 g for 5 minutes and supernatant was used for colony Polymerase Chain Reaction (PCR).PCR-Randomly Amplifi ed Polymorphic DNA (PCR-RAPD) was performed as described earlier using 1 μl of supernatant as a DNA template 16 .The class 1 integron element integrase gene (intI1) and ESBL genes (bla CTX-M , bla TEM-1, and bla SHV ) were amplifi ed using the protocol published 17 .Briefl y, the reaction mixture contained 0.5 M each primer, 250 M each deoxynucleoside triphosphate, 1U Taq DNA polymerase (Finzymes), 2.5 μl of 10X PCR buff er, and 100-500 ng of total genomic DNA in a fi nal volume of 25 μl.Amplifi cation was performed in a Perkin-Elmer thermocycler 2400 model for 5 min at 95°C, followed by 30 consecutive cycles of 1 min at 95°C, 1 min at 50°C for bla TEM gene, 55°C for bla SHV , 58°C for bla CTX-M gene, and 1 min at 72°C, with a single fi nal extension step of 10 min at 72°C.PCR products were separated by electrophoresis in a 1.5 percent agarose gel and stained with ethidium bromide.

Results
The isolate C316709 and C109 were sensitive to ofl oxacin, ciprofl oxacin and norfl oxacin while isolate C3409 was resistant to all eleven antibiotics tested (Table 1).The IntI1 was amplifi ed from two isolates (C3409 and C109).All isolates were positive for bla CTX-M and an isolate C3409 also harbored bla TEM-1 .bla SHV was not amplifi ed from all isolates.The PCR-RAPD genotyping of these isolates showed three diff erent genotypes (a, b and c) and they were not clonally related (Table 1 and Fig. 1).

Thapa B et al
Despite the fact that Citrobacter spp.infection is prevalent in Nepal, it is unfortunate that no systematic molecular studies have been conducted, however the isolation of Citrobacter spp.have been described 2,8,9 .In this study, we have shown the emergence of C. freundii carrying class 1 integron element and bla CTX-M and bla TEM- 1 ESBL genes for the fi rst time in Nepal.The present study has also identifi ed C. freundii strain simultaneously harboring bla CTX-M and bla TEM-1 which has also been described in India 18 .These Indian isolates also carried bla SHV and bla ampC along with bla CTX-M and bla TEM-1 .C. freundii carrying bla CTX-M has also been described elsewhere 7 .Enterobacteriaceae carrying class 1 integron element have been described, however the reports describing the presence of this mobile genetic element in Citrobacter is scarce.We have also identifi ed C. freundii carrying this mobile genetic element.Most of the ESBLs are carried in this mobile genetic element 19 and the carriage of ESBL genes in this element was not investigated.Citrobacter spp.infection has frequently been increasing as a cause of serious concern, especially for neonates and immunocompromised adults.The increasing antibiotic resistance is also noteworthy for C. freundii.
The neonatal mortality of Nepal was 33/1000 live births in 2006 and the preliminary fi ndings of National Demographic and Health Survey 2011 has shown no diff erence in NMR 20 .To address maternal health, Ministry of Health and Population, Government of Nepal has started "AMMA" program, which aims to motivate institutional delivery to decrease maternal mortality.In light of emergence of class 1 integron element and ESBL harboring C. freundii, government's eff ort of advocating institutional delivery and saving maternal lives will position neonates at increased risk of neonatal morbidity and mortality if infection prevention and control policies and practices are not in place.Furthermore the spread of mobile genetic element and resistance genes will help in emergence of multidrug resistant pathogens and might pose challenge for saving newborns.
In conclusion, this study has highlighted the spread of oligoclonal population of C. freundii neonatal nosocomial infection in NICU, Nepal and has also documented the spread of bla CTX-M and bla TEM-1 and class 1 integron element carrying C. freundii in Nepal.

Fig1:
Fig1: PCR-RAPD patterns of C. freundii isolates.The lanes have been marked with the respective isolates.M, Molecular weight marker (1 Kb+ Invitrogen, USA).Lane 1, pattern a; lane 2, pattern b and lane 3, pattern c.The PCR-RAPD was performed in Department of Microbiology, Siriraj Hospital, Bangkok, Thailand.

Table 1 :
Characteristics of Citrobacter freundii isolates