Blood Stream Infections caused by Non-Fermenting Gram Negative Bacilli, Clinical Correlation, MIC for Colistin, Gene Detection

To study the risk factors and outcome of blood stream infection caused by non-fermenting gram negative bacilli (NFGNB) and their pattern of antibiotic susceptibility and genes. We included sepsis cases with blood culture positive for NFGNB. MIC for colistin was determined by broth microdilution method. Multiplex PCR was used to detect BlaIMP, BlaVIM, BlaKPC, BlaNDM-1 genes in cephalosporin and carbapenems resistant Acinetobacter spp. isolates. Out of 4,664 cases of sepsis, 50 (1.07%) were positive for NFGNB. Acinetobacter spp. 29 (58%) was the predominant isolate, of which 16 (55.17%) isolates were resistant to cephalosporins and carbapenems. We detected BlaKPC and BlaNDM-1 genes in two of these isolates. We did not detect BlaIMP, BlaVIM, BlaKPC and BlaNDM-1 genes in any other NFGNB isolates. Majority of the strains of Pseudomonas spp. showed sensitivity to all the antibiotics tested. NFGNB sepsis patients with respiratory illness correlated well with fatal outcome (p <0.05; OR 21). More numbers of Acinetobacter spp. sepsis cases had fatal outcome (p <0.05; OR 12.83). NFGNB sepsis patients with respiratory illness and those which yielded Acinetobacter spp. correlated positively with fatal outcome. We detected BlaKPC and BlaNDM-1 genes in two strains of drug resistant Acinetobacter spp.


INTRODUCTION
Gram negative bacilli which are nonfermenters (NFGNB) are a heterogeneous category of microorganisms which do not have the ability to ferment sugars. NFGNB, which were considered as contaminants in the past, have emerged as important major pathogenic organisms responsible for causing a variety of infections in recent years 1 . NFGNB cause opportunistic healthcare-associated infections. They are commonly found on skin of healthcare-workers, instruments such as ventilator machines, humidifiers and mattresses [2][3][4] . Among NFGNB Burkholderia cepacia, Pseudomonas aeruginosa, Acinetobacter baumanni complex, Alcaligenes faecalis, Sphingomonas paucimobilis and Stenotrophomonas maltophilia are some pathogens known for causing health care associated bloodstream infections 5 .
Multidrug resistance (MDR) property of NFGNB is the reason that facilitated the way for the reconsideration of previously used antibiotic colistin into clinical use. For the treatment of infections caused by multi-drug resistant gramnegative bacilli (MDRGNB), colistin may have crucial and reliable role as potent antibiotic. 6,7 The clinical use of colistin was restricted because of reports of serious nephrotoxicity and discovery of less toxic antibiotics 6,7 . So we intend to study the antibiotic sensitivity pattern of NFGNB from sepsis cases and correlate with the clinical condition of the patient. We also determine MIC of Colistin and also detect Bla IMP , Bla VIM , Bla KPC , Bla NDM-1 genes in cephalosporin and carbapenems resistant Acinetobacter spp. isolates.

MATERIAlS AND METhODS
It is a prospective time bound study conducted from (1 st November 2018 to 30 th April 2019) at the Microbiology laboratory of a tertiary care center. Sepsis cases with blood culture positive for NFGNB were included in the study. Blood culture samples which did not grow NFGNB and samples received on days other than the duration specified were excluded. Non random sampling strategy was used. Clinical data was taken from the records section of the hospital. Institutional ethics committee clearance has been taken for the project.
Bac T/Alert bottles with patient's blood received in microbiology laboratory, for blood culture were processed as per protocol, in Bac T/Alert 3D system. From the positive bottles, the subcultures were done on MacConkey agar, Blood agar and Chocolate agar. The identification of NFGNB up to the species level was done by VITEK ® 2 Compact(C) system using the Gramnegative Identification (GN-ID) 21341 card, and antibiotic sensitivity testing (AST) was done with AST 281 card. Both the Bac T/Alert 3D system and VITEK ® 2 Compact (C) system were from bioMerieux company (North Carolina, USA), MIC for colistin by broth micro dilution method MIC for colistin by broth microdilution method was carried out as per CLSI guidelines. 8 A 100 mL stock solution of colistin sulfate (Sigma Aldrich, USA) of concentration 256 μg/mL was prepared according to the directions provided by the drug manufacturer. Two fold dilutions of the antibiotic were made to get a final dilution of 128, 64, 32, 16, 8, 4, 2, 1, 0.5 & 0.25 μg per ml of colistin respectively in wells 1 to 10 of microtitre plate.
Inoculum was prepared from 4 to 5 isolated colonies of similar colony morphology grown over night (18-24 hours) on Mac Conkey's agar. The colonies were inoculated into cation adjusted Mueller Hinton broth (CAMHB), kept at 37°C for 4 to 6 h to get log phase growth and adjusted to 0.5 McFarland (1 x 10 8 CFU/ml). This broth was repeatedly diluted to get the final concentration of bacteria as approximately 5 x 10 4 CFU/ ml. Now 10μl of inoculum was pipetted into wells 1 to 11. Well 11 acted as the growth control and 12 th well as broth sterility control with sterile CAMHB. The microtitre plates were incubated for 12-18 h at 37°C. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were run as controls. The lowest concentration of antibiotic at which there is no visible growth of organism was taken as MIC endpoint. According to CLSI, ≤ 2 µg/ml and ≥ 4 µg/ml were considered as the susceptible and resistant break points respectively. Multiplex PCR 9,10 The DNA was extracted according to CDC protocol using boiling method. The primer sets were purchased from sigma Aldrich Pvt. Ltd ( Table 1). The master mix was prepared by adding ready mix Taq PCR reaction mix with MgCl 2 (Califormia, USA), in which the respective primer sets i.e, Forward (Fˈ) as well as Reverse (Rˈ) and the testing DNA were added and this master mix was subjected to amplification. Amplification was done with following cycling conditions;10 min denaturation at 95°C, then 35 cycles of (45s denaturation at 94°C, 45 s primer annealing at 54°C, and 50 s primer extension at 72°C). The last cycle, an additional 7 min extension step at 72°C in Rotor gene -Q (Qiagen, germany) and the products were kept at 4°C. The amplified product was detected by using 2% agarose gel electrophoresis. K.pneumoniae ATCC BAA 1705 and K.pnemoniae BAA 1706 were used as positive and negative control. Clinical isolates known to be positive for Bla IMP , Bla VIM Bla NDM-1 genes were also used as controls.

Statistical analysis
Statistical analysis was performed using SPSS version 16.0. Multiple logistic regression analysis was done for correlation of risk factor and fatal outcome. Chi Square test was also used to correlate other variables.
In a study on resistance of NFGNB isolated from blood cultures from an emergency hospital in Brazil found that from 87(100%) isolated and analyzed strains, 11(13%) were NFGNB. Acinetobacter spp. was most often occurring organism (55%) succeeded by Pseudomonas spp. (18%), Stenotrophomonas spp. (18%) and Burkholderia spp. (9%). Acinetobacter spp. were resistant to gentamycin, meropenem, imipenem, amikacin, ciprofloxacin, ceftazidime and ceftriaxone. Pseudomonas spp. was sensitive to gentamycin, meropenem, imipenem, amikacin, levofloxacin, norfloxacin, ceftazidime and cefepime. All Stenotrophomonas spp. were resistant to levofloxacin and chloramphenicol. Burholderia spp. were sensitive to the antibiotics tested 11 . A study showed A.baumannii isolated from sepsis cases was highly resistant to aminoglycosides in comparison to other Acinetobacter spp. P. aeruginosa strains were sensitive to all the aminoglycosides tested. 12 We have similar results.
Rate of isolation of NFGNB from blood vary in studies conducted from various places from 0.7 to 20.8 13,14 . In a study by Grewal US et al isolation rate of NFGNB was 11.6%. Commonest NFGNB isolated in the study was P. aeruginosa (87.96%), followed by A. baumannii (7.87%). 24.2 % of P. aeruginosa strains were identified as multi drug resistant (MDRPA). About 100% of the MDRPA isolates were found to be susceptible to polymyxin B. However, 60.9% of the MDRPA isolates in their study showed resistance to imipenem, which is usually the first therapeutic choice for treating the infections caused by them. Isolates of A. baumannii in their study showed maximum susceptibility to imipenem (88.2%), followed by cefoperazone + sulbactam (76.4%). Maximum resistance was shown to aztreonam (11.7%). 11 isolates of A. baumannii showed multidrug resistance (MDRAB) in the study. Isolation of multi drug resistant P. aeruginosa and multi drug resistant A. baumannii in the study raised the concern of emerging antibiotic resistance in this group 14 .
In an another study on predominance of NFGNB and sensitivity pattern in Tamil Nadu, found that out of 5052 clinical samples, 517 (10.2%) samples were NFGNB. Acinetobacter and Pseudomonas spp. were the most frequently found organisms. Colistin and imipenem showed maximum sensitivity for all clinical samples. Highest resistance was seen with gentamycin ceftazidime and ciprofloxacin 15 .
An antibiotic trend analysis over a period of seven years in patients suffering from nosocomial infections showed elevated levels of resistance to frequently used antibiotics and hence a significant rise in colistin use especially in patients who were critical 16 .
In a retrospective study factors deciding the existence of patients with Acinetobacter baumannii bacteremia were evaluated. Relevant details of hospitalized patients who developed Acinetobacter baumannii bacteremia were collected in Beirut between 2010 and 2015. Presence of diabetes mellitus, large amount of steroids, ventilator use, early treatment with tigecycline and colistin, critical care unit and septic shock correlated with worse result by univariate analysis. Multivariate analysis showed significant correlation with large doses of steroids and septic  17 . In our study there were no positive correlation among death and diabetes mellitus. But as in previous studies we got positive correlation between death and underlying respiratory illness like, VAP, COPD, bronchial asthma and pneumonia. We also found that mortality was associated with sepsis in Acinetobacter spp.
In a study on carbapenem resistant strains of coliforms isolated from cases of blood stream infection bla NDM-1 & bla KPC genes were detected. They found that 16 isolates harbored bla NDM-1 , and bla KPC gene was not detected in any of the isolates 9 . In a study period of 2008 and 2012, 166 uropathogens isolated from cases with complicated UTI, 34(43.6%) isolates had bla VIM gene, 5(6.4%) had bla IMP , bla NDM-1 and bla KPC genes were absent. Among the isolates from 2012, 47(53.4%) had bla NDM-1 gene 19(24.4%) had bla VIM , one (1.1%) had bla IMP and bla KPC gene was absent 10 . In the present study one each of Bla KPC and Bla NDM-1 genes were detected in two strains of drug resistant Acinetobacter spp. Further studies with a larger sample size may give more conclusive results.

CONClUSION
Rate of isolation of NFGNB from blood from sepsis cases was 1.07%. Most commonly isolated bacteria were A. baumannii and P.aeruginosa. NFGNB sepsis patients with respiratory illness and those which yielded Acinetobacter spp. correlated positively with fatal outcome. Most isolates of Acinetobacter spp. were sensitive to amikacin and tigecycline. Sixteen isolates of Acinetobacter spp. were resistant to cephalosporin and carbapenems. Most strains of Pseudomonas spp. were sensitive to all antibiotics tested. Only one isolate was resistant to carbapenems and cephalosporin. All strains of Acinetobacter spp. were sensitive to colistin and Two isolates of Pseudomonas species were resistant to colistin. One each of Bla KPC and Bla NDM-1 genes were detected in two strains of drug resistant Acinetobacter spp. Further studies with a larger sample size may give more conclusive results.

ACKNOWlEDGMENTS
We are extremely grateful to our institution Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India, for providing us with required infrastructure and all the materials for the conduct of this research project.