Characterization of Antibiotic Resistance Determinants of extended Spectrum β-lactamases Producing Enterobacteriaceae from Cancer Patients in South india

Patients with malignancy are highly prone to infections by extended spectrum beta-lactamases producing Enterobacteriaceae (eSBl-Pe). Knowledge on local resistance profile and resistance genes is essential to decide empirical drug. Hence, the study aims to find the resistance profile and the resistance genes of eSBl-Pe from cancer patients. 172 oxyimino-cephalosporins resistant enterobacterial isolates from clinical specimens of cancer patients were obtained. Study isolates were speciated by conventional biochemical methods. Resistance to antibiotics was detected by disc diffusion method. Phenotypic detection of eSBls was performed as stated in ClSi guidelines. Genotypic characterization of resistance determinants of eSBl-Pe was done by PCR. Among 172 enterobacterial isolates, 151 (87.7%) were eSBl producers. E. coli (67.5%) was the major species producing eSBl enzymes followed by K. pneumoniae (27.8%). Antibiotic susceptibility pattern showed lowest resistance to imipenem 11.2%, and netilmicin 13.9%. 72% of eSBl-Pe was found to be Multidrug-resistant. Among eSBl genes, bla CTX M gp-1 (83.4%) was dominant followed by bla TEM (32.4%) and bla SHV (27.8%). 36% of the isolates were found to be positive for more than one eSBl gene. High level of plasmid encoding quinolone resistance genes (64.2%) was identified in eSBl-Pe. low levels of plasmid mediated AmpC gene (15.8%) and 16S rRNA genes (9.2%) were found in eSBl-Pe. the predominant eSBl encoding gene belongs to bla CTX M group 1. High proportion of eSBl-Pe was found to co-harbor PMQR genes. eSBl-Pe had highest sensitivity for imipenem and netilmicin.


iNtRODuCtiON
Patients with malignancy are more susceptible to bacterial infections, as they are often immunocompromised due to malnutrition and various treatment modalities.Infection in cancer patients increases the hospital stay, financial burden, morbidity and mortality. 1,2nterobacteriaceae accounts for approximately 65%-80% of Gram negative bacterial infections in cancer patients. 3Resistance in Enterobacteriaceae due to Extended spectrum β-lactamases (ESBLs) are reported increasingly and the organisms producing these enzymes has spread worldwide and considered as important pathogens in community and hospital settings. 4,5ESBLs producing Enterobacteriaceae (ESBL-PE) is a leading organism causing infection in cancer population and considered as major threat since it leads to serious illness in those patients. 6ide usage of oxyimino-cephalosporins as therapeutic options for enterobacterial infections led to the appearance of ESBLs. 5ESBL enzymes hydrolyzes penicillins, cephalosporins except cephamycins.ESBL genes are encoded by plasmid that facilitates its dissemination among different Enterobacteriaceae species. 7The major types of ESBLs were from TEM, SHV, and CTX M families. 8la CTX-M-15 gene has emerged all over the world, also common in nosocomial and community acquired infections. 9ESBL producing Enterobacteriaceae are often non-susceptible to fluoroquinolones, co-trimoxazole and aminoglycoside, due to coexistence of different resistance determinants on same plasmid and so exhibit the Multidrugresistance phenotype and limits the treatment options. 10Although several studies of ESBL-PE species from hospital and community settings have been reported, very few data are available on ESBL-PE isolates from cancer population.The few reports on ESBL-PE are pertaining to bloodstream infections and there is hardly report on antibiotic resistance determinants of ESBL-PE from diverse infections in cancer patients.It is significant to identify the sensitivity pattern and resistance genes of Multidrug-resistant pathogen like ESBL-PE to aid appropriate choice of empirical therapy for infection in this high risk population.
Hence the present study was designed to find antibiotic susceptibility pattern and characterize the various antibiotic resistance genes of ESBL-PE isolates from cancer patients.

Study isolates
172 oxyimino-cephalosporins resistant Enterobacteriaceae were collected from Cancer care hospital, Chennai, Tamil Nadu, South India during the period commencing from February 2016 to May 2017.The collected isolates were obtained from various clinical specimens (urine, pus, sputum etc,) of cancer patients.

identification and speciation of study isolates
Isolation of the study isolates from the clinical specimens was done in hospital according to standard microbiological procedure.Only the pure culture isolates collected from the hospital were used in the study.All the 172 isolates were identified and speciated by using standard techniques such as Gram staining, studying colony morphology on MacConkey agar (Hi-Media Labs, Mumbai, India), oxidase test, catalase test, nitrate reduction, O/F test, TSI, IMViC ( Indole, Methyl Red, Voges Proskauer and Citrate), urease production, phenylalanine deaminase test, motility tests and sugar fermentation tests.

Phenotypic confirmation of esbls
Phenotypic confirmation of ESBL production was done by disc diffusion test as stated in CLSI guidelines. 11The ceftazidime (CAZ-30 μg) and Cefotaxime (CTX-30 μg), discs with and without clavulanic acid (10 μg) (Hi-Media Labs, Mumbai, India), were placed 30 mm apart on a lawn culture of the test isolate (0.5 McFarland opacity)and incubated for 24 hours at 37°C.Bacterial isolate was considered ESBL positive if ≥ 5mm increase in zone diameter of the clavulanic acid combination disc was observed than the disc without clavulanic acid.

Molecular detection of eSBl encoding genes and associated resistant genes
DNA extraction was carried out by boiling lysis method.Colonies of study isolates were inoculated in Luria-Bertani broth and incubated overnight.Then the broth was centrifuged and 200μl sterile water was added to the pellet; kept in dry bath at 100°C for 10 mins; immediately kept in -20°C deep freezer followed by centrifugation.Multiplex PCR detection of bla CTXM gp-1, bla CTXM gp-2, bla CTXM gp-8, bla CTXM gp-9, bla CTXM gp-25 genes was done. 12The presence of bla TEM and bla SHV were identified by the multiplex PCR according to the published methods. 13[16][17]

Statistics
GraphPad QuickCalcs were used to perform statistical analysis.Chi-square test was used to calculate associations between antibiotic resistance and its genes.A p-value of < 0.05 was considered statistically significant.
Non-ESBL producing isolates showed highest resistance to fluoroquinolones and piperacillin.(Refer Figure)

Genotypic characterization of eSBl and other resistant genes
Among the ESBL genes detected, bla CTX M gp1 was the predominant ESBL type found in 83.4% (126/151) of the isolates followed by bla TEM 32.4% (49/151) and bla SHV 27.8% (42/151).36% of the test isolates were found to be positive for more than one ESBLs encoding genes.The bla CTXM gp2, bla CTXM gp8, bla CTXM gp9, and bla CTXM gp25 genes were not found in any of the ESBL-PE.64.2% of the ESBL producing strains harbored one or two PMQR genes, qnrA was not found on any of the isolates, 15.8% of the isolates were found to carry plasmid-mediated AmpC genes and 9.2% of the isolates were associated with 16SrRNA methylase gene (armA, rmtB).All the isolates were negative for rmtC gene (Refer Table 4).The statistical analysis revealed that the association between the antibiotic resistance and its determinants was not significant.

DiSCuSSiON
In this study, it has been observed that 87.7% of the oxyimino-cephalosporin resistant enterobacterial isolates from cancer patients were carrying ESBL encoding genes.This is consistent   In this study, E. coli was the leading ESBL-PE isolate followed by K. pneumoniae and the chief sources of these isolates were urine samples.This is in accordance with the report of Barta et al., and Jiang et al. 18,19 Earlier findings state that ESBL-PE is one of the leading pathogens causing bloodstream infections in cancer patients. 20,21This is contrary to the findings of our current study which showed only 2% of ESBL-PE isolates were from blood samples.The findings of the present study suggests that other sites of infections viz., urinary tract, respiratory tract, gastro-intestinal tract, surgical site, wound are also important sources of ESBL-PE infections.Although, there are several reports available for bloodstream infections in cancer patients, very few reports are available on ESBL-PE infections from other clinical sites.
In this report, ESBL production was phenotypically negative in 7% of PCR positive ESBL isolates.A surveillance study from India reported 40% PCR positive ESBL isolates were undetectable phenotypically.Production of multiple β-lactamases can mask the inhibitory effect of clavulanic acid and may make ESBLs phenotypically undetectable. 22he antibiotic susceptibility testing of the current study revealed high level of non-susceptibility to non-beta-lactam drugs.ESBL-PE isolates showed higher resistance to fluoroquinolones and co-trimoxazole as previously reported by Aldrazi et al., and Mahamat et al. 23,24 Resistance to other classes of antibiotics among ESBL-PE is worrisome because it further limits the empirical therapeutic options.ESBL-PE showed less resistance to aminoglycosides particularly netilmicin.The resistance rates of amikacin was 38% and of gentamicin 36% unlike other reports.A study from Spain reported 13% of gentamicin resistance and nil amikacin resistance among ESBL-PE which is much lower than the current study. 25nother study has reported 70.2% of gentamicin resistance, 67% of netilmicin resistance and 18% of amikacin resistance in ESBL-PE which is contrary to the current report. 24These variations may be due to different geographical locations and study period.
Carbapenems are the generally preferred drugs for the treatment of ESBL-PE infection and it has lower failure rate in treating these infections compared to cephalosporin and fluoroquinolones. 26In the current study, ESBL-PE showed high susceptibility to carbapenems.They showed higher susceptibility to imipenem than meropenem which is in concordance with previous report from India. 22Even low rate of carbapenem resistance among ESBL-PE is of major concern and hence it should be used with caution.
In the current study, increased non-susceptibility was observed for ampicillin, piperacillin, and 3 rd generation cephalosporins and this is due to the production of ESBLs and other betalactam hydrolyzing enzymes.
In the current study, the predominant ESBL encoding gene among the enterobacterial isolates is bla CTXM group1 (83.4%) followed by TEM (37.4%) and SHV (27.8%).Several studies have reported similar findings of high levels of bla CTXM genes among Enterobacteriaceae. 24A study from India by Ensor et al., has reported high rate of bla CTXM (73%) among cephalosporin resistant Enterobacteriaceae. 27ESBL genes bla CTX-M15 belongs to bla CTX-M group1 and has disseminated worldwide and is widely reported as predominant ESBL gene among Enterobacteriaceae. 9In the present report, bla TEM was found in 37.4% ESBL-PE isolates; contrary to previous reports from India.A study by Jena et al, reported high proportion of bla TEM 96.42% in ESBL-PE 27 and another study by Ray et al., has also reported bla TEM as the most common ESBL among E. coli and K. pneumoniae. 22la SHV gene was detected in less proportion (27.8%) in the current study and this is similar to the earlier study from India which documented lower rates of bla SHV (6% and 21%) among E. coli and K. pneumoniae isolates.22 Co-occurrence of ESBL genes were observed in the present study and the same has been reported in the earlier studies.22,24,28 Findings of the current study demonstrate the co-harboring of various resistant determinants    (10)  qnrS (3) rmtB (2) bla SHV (3)  aac (6′)-Ib-cr (41) bla CTX-M gp-1+ qnrB+aac(6′)bla TEM (17)  Ib-cr (13) bla CTX-M gp-1+ qnrS+aac (6′)bla SHV (10)  Ib-cr (2) bla CTX-M gp-1+ bla TEM +bla SHV (2)
The study has certain limitations.The study data was from single centre, hence it may not depict epidemiology of different geographical locations.More clinical details of the cancer patients such as previous exposure to antibiotics and other co-morbidities are unavailable.

CONCluSiON
To the best of our knowledge, this is the first report regarding the antibiotic resistance determinants of ESBL-PE from cancer patients from our geographical location.The study data showed high prevalence of ESBLs among Enterobacteriaceae from cancer patients.The predominant ESBL encoding gene was bla CTX M group 1. High proportion of ESBL-PE was found to co-harbor PMQR determinants and co-existence of AmpC and 16SrRNA methylase genes were also observed.Most of the ESBL-PE isolates showed Multidrug-resistant phenotype with few treatment options.Thus, the judicious usage of antibiotics and surveillance program should be implemented for cancer patients to prevent spread of ESBL-PE.Imipenem and netilmicin have highest sensitivity for ESBL-PE and can be considered for the treatment in our region.Further studies on prevailing resistant pattern from different regions of India are required to affirm the appropriate empirical therapeutic options.

Figure .
Figure.Non-susceptibility pattern of ESBL genes positive and negative isolates

table 2 .
Clinical characteristics of cancer patients infected by ESBL-PE

Table 2
shows the clinical details of the patients infected by ESBL-PE.n indicates no.of ESBL isolates

table 3 .
Species distribution of ESBL producing isolates and its source