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Bacteriology & Mycology: Open Access

Research Article Volume 11 Issue 1

Comparative study of C-reactive protein and complete blood count in cancer and non-cancer patients followed by antibiogram analysis of isolated bacterial pathogens

Asif Iqbal,1 Dr. Riaz Muhammad,2 Bakht B Khan,3 Aleesha Jamshed,4 Muhammad IK Rehman,2 Sawaira Iqbal5

1Department of Microbiology, Hazara University Mansehra, Pakistan
2Department of Microbiology, Abasyn University Peshawar, Pakistan
3Professor Peshawar Medical College, Pakistan
4Departments of Environmental Sciences, COMSATS University, Abbottabad Campus, Pakistan
5Department of botany, GPGC Nowshera kpk Pakistan

Correspondence: Asif Iqbal, Department of Microbiology, Hazara University Mansehra, Kp Pakistan-21300

Received: December 30, 2022 | Published: January 11, 2023

Citation: Iqbal A, Muhammad R, Khan BB, et al. Comparative study of C-reactive protein and complete blood count in cancer and non-cancer patients followed by antibiogram analysis of isolated bacterial pathogens. J Bacteriol Mycol Open Access. 2023;11(1):1-4. DOI: 10.15406/jbmoa.2023.11.00334

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Abstract

Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. This study focuses on the analysis of blood cell and their proteins followed by antibiotic susceptibility and ESBL detection in Cancer and non-Cancer patients. In this study, a total 200 blood and urine samples were screened out for bacteria especially ESBL producing bacteria. Out of the tested samples, different bacterial pathogens were identified and among the isolates E coli were (13.33%), S. aureus (11.66%), P. aeruginosa (11.66%), salmonella (10%), bacillus spps (9.16%), Enterobacter spps (8.33%), Mycobacterium Spps (7.5%), S. Pyogene (7.5%), H. pylori (6.66%), Klebsiella spp. (5.83%), S. epidermidis (4.16 %) and Shigella was (4.16%). The ESBL producing bacteria among the isolated 120 bacterial species were only 14 bacterial isolates are ESBL producers which are E. coli (06), P. aeruginosa (04). Enterobacter spp (04), Klebsiella spp (03) and Shigella were only (01). The 100 blood samples are also analysis through CRP test in which 50 blood samples taken from cancer patients and 50 from non-cancer patients. In total 50 cancer sample CRP analysed that 21 patents have the Lowest (0.92 mg/L), 08 was (0.92-1.93 mg/L) 11 was (1.94-3.69 mg/L) and10 patients were on the Highest (>3.69 mg/L) risk. In 50 non-cancer blood sample the CRP level are 09 individual were (0.92 mg/L), 11 individual (0.92-1.93 mg/L), 25 individual (1.94-3.69 mg/L) and 03 patients were Highest (>3.69 mg/L) risk. Maximum resistivity (91%) was showed by penillion (P) and maximum sensitivity (78%) was showed by rifampicin (RD) against isolated bacterial pathogens. It is recommended that Continuous ESBL screening and supervision are necessary at hospital settings to observe and develop approaches for observing and controlling the spread of ESBL generating bacteria.

Keywords: blood, cancer, bacteria, pathogens, CRP, ESBL

Introduction

The most important part of cancer research over the past numerous years have been complete on the comprehensive study of the mechanisms of malignancy etiology.1 Cancer is an extremely difficult progression. Carcinogens (cancer causing agents) such as viruses and some of the 200 chemicals found in cigarette smoke are processed in the body and then cause modifications to DNA.2 This stage of carcinogenesis3 is known as initiation. In the subsequent stage-promotion cells that carry damaged or mutated DNA may undertake alterations that allow them to progress into a cancer. Many factors modify these processes, such as hormones, genetics, body weight, and the many constituents present in the diet. CRP was discovered nearly 70 years ago by scientists exploring the human inflammatory response.4 White Blood Cells (WBC) are the main and important type of blood cells which circulate throughout the blood vessel and the lymphatic system. These are very important and defensive cell of immune system, because WBCs perform very important role in defence against microbial antigen.5 CRP is a significant acute phase complementary system associated protein which is present in normal serum of humans and produced by liver due to the macrophage which release cytokines (IL-1, IL-6, IL-8, TNF etc.) when bacteria is engulf by macrophage.6 CRP is the first protein of complementary system which binds to antigen.7 The bacteria present in various types of cancer patients were Salmonella, Shigella, Campylobacter, H. pylori, Mycobacterium tuberculosis, Escherichia coli, Listeria, Actinobacter baumannii, Streptococcus pyogene, Streptococcus mutant, Salmonella, Staphylococcus aureus, Vibrio cholera and Yersinia etc.8 These bacterial species cause different opportunistic infections in cancer patients due to the compromised immune system and many other reasons. Among β-lactamases, Extended-Spectrum Beta–Lactamases (ESBLs), Ampciline C-type β-lactamases, in addition to carbapenemase are of abundant apprehension.9 Antibiotic resistance is rapidly increase in that regions where the people have no proper hygiene and immoral use of antibiotics.10 One main reason is the rapid traveling of people from one country to another for different purposes especially from that part of the world where resistant bacteria exist especially ESBL generating Bacteria are present in some parts of Asia, Africa and South America.11 The current paper focuses on the analysis of blood cell and antibiotic susceptibility of bacterial pathogens in Cancer and non-Cancer patients.

Methodology

The current research work will be carried out in MRL in the Department of Microbiology and Biotechnology, Abasyn University Peshawar and in Microbiology and Hematology Laboratory of Pathology Department HMC from March 2017 to December 2017.

Total 200 blood and urine samples were collected from cancer patients of various types (Liver, Lungs, Blood, Skin and Stomach cancer) and non-cancer patients within one to two month. The samples were collected from different hospital located at Peshawar City i.e. mostly from HMC, IHP and LRH Peshawar.

CBC and CRP

Blood samples each with 4.5ml blood were collected in green EDTA tubes then the samples were first mixed. Then the blood sample was run in Sysmex and cell dyne ruby (CDR) blood counting apparatus.12 The C-reactive protein (CRP) test was performing accordingly.13,14

Urine analysis

Total 100 Urine sample were collected and out of these 50 samples was taken from cancerous patient and 50 from non-cancer patient. Then the physical, chemical and Microscopic analysis was conducted for the evaluation of patient Blood cells and for the presence of bacteria in patients’ Urine.15

Identification of isolated bacteria

The different culture Media was used i.e., Nutrient agar, blood agar, CLED agar, MacConkey agar, Eosin Methylene Blue (EMB), Salmonella, Shigella Agar (SSA). Pathogenic bacteria were isolate though serial dilution method on nutrient agar. The pathogenic bacteria form a colony; from colony different bacterial species were isolated according to their morphology, Gram’s reaction and biochemical test.16

Antibiotic susceptibility testing

Disc diffusion assay          

Antibiotic sensitivity was done through Kirby-Bauer disc diffusion technique. Standard protocols mentioned in Clinical and Laboratory Standards Institute (CLSI) 2015 was followed. Different antibiotics (Amoxicillin, cyclosporine, Vancomycin, Gentamycin, Tazobactum, Cefotaxime, Ciprofloxacin, Rifampicin, Doxycycline, Chloramphenicol, Polymaxin-B, Carbapenam, tetracycline, etc.) of various concentrations was evaluated. According to the type of isolated bacteria various types of antibiotics was tested against isolated pathogenic bacteria according to the Clinical and Laboratory Standards Institute (CLSI) 2015 protocol.

In the disk diffusion test, the bacterial isolate is inoculated uniformly onto the surface of an agar plate and a filter disk impregnated with a standard amount of an antibiotic is applied to the surface of the plate, resulting in a gradient of the antibiotic surrounding the disk. Following incubation, a bacterial lawn appears on the plate and zones of inhibition of bacterial growth would be present around the antibiotic disk. The test is performed under standardized conditions hence the size of the inhibition zone is dependent on the degree of sensitivity of the microorganism to the antibiotic.17

Detection of ESBL producing bacterial isolates

A 0.5 MacFarland’s suspension of every single insulates was spread on a Muller – Hinton agar (MHA) plate Ceftazidime (30 μg) Ceftazidime and clavulanic acid (30 μg/ 10 μg) discs was positioned aseptically on the agar having plate. The space of approximately 15mm was retained in the middle of the two discs (edge to edge) and the cultures were incubated at 37 C overnight. The observation was equal or greater than 5mm rise in the zone diameter for the antimicrobial agent which was tested in combination with clavulanic acid, against its zone thickness when tested only, confirmed the occurrence of Extended Spectrum Bata Lactamase (ESBL) manufacture by the bacteria.18

Results and discussion

Present study was carried out to determine the current scenario of CRP, CBC, and bacteria resistance present in cancer and non-cancer patients. In this study, a total 200 blood and urine samples were screened out. Out of the tested samples, different bacterial pathogens were identified and among the isolates E coli were (13.33%), S. aureus (11.66%), P. aeruginosa (11.66%), salmonella (10%), bacillus spps (9.16%), Enterobacter spps (8.33%), Mycobacterium Spps (7.5%), S. Pyogene (7.5%), H. pylori (6.66%), Klebsiella spp. (5.83%), S. epidermidis (4.16 %) and Shigella was (4.16%). Similarly a study also by Helde-Frankling et al.,19 isolated bacteria in various varieties of cancer are Salmonella, Shigella, Campylobacter, H. pylori, Mycobacterium tuberculosis, Escherichia coli, Listeria, Actinobacter baumannii, Streptococcus pyogene, Streptococcus mutant, Salmonella, Staphylococcus aureus, Vibrio cholera and Yersinia. Our study shows the some indication of viral and fungal spps as well. However we don’t search out viral and fungal infections. The outcomes of Sengar et al.20 are somehow in confirmatory with our results where the frequency of Gram-Negative Bacteria remained (51.59%), in which E. coli (12.79%) Pseudomonas species were (9.71%) Salmonella (8%), and gram positive (40.72 %) in which staph aureus were (9.23%) Bacillus spps (9.16%) respectively. However the outcomes of Parveen et al.21 also reported the Spiral or curved bacilli isolates and genus Campylobacter (Table 1 & 2).

Total samples

Negative samples

Total positive bacterial isolates

Total bacterial isolates in cancer samples

Total bacterial isolates in non-cancer samples

Total bacteria isolates from blood samples

Total bacteria isolates from urine samples

200

80

110

51

59

30

80

Table 1 Positive sample distribution of bacterial isolates in various samples

S no.

Name of bacteria isolated

Count

Percentage (%)

ESBL positive isolates

01

E. coli

16

13.33

06

02

S. aureus

14

11.66

Nil

03

P. aeruginosa

14

11.66

04

04

Salmonella

12

10

Nil

05

bacillus spp.

11

9.16

Nil

06

Enterobacter spp.

10

8.33

04

07

Mycobacterium Spp.

09

7.5

Nil

08

S. pyogene

09

7.5

Nil

09

H. pylori

08

6.66

Nil

10

Klebsiella spp.

07

5.83

03

11

S. epidermidis

05

4.16

Nil

12

Shigella

05

4.16

01

Total

120

100

18

Table 2 Percentage distributions of various bacterial pathogens identified in both cancer and non-cancer samples and ESBL bacterial Isolates

In the present research work we also detect ESBL producing bacteria among the isolated 120 bacterial species. Out of these only 14 bacterial isolates are ESBL producers which are E. coli (06), P. aeruginosa (04), Enterobacter spp (04). Klebsiella spp (03) and Shigella were only (01). The ESBL producing isolates were detect though double disk diffusion assay in which Ceftazidime (30 μg) Ceftazidime and clavulanic acid (30 μg/ 10 μg) discs were used. The Findings of Bos et al.22 also isolated ESBL producing bacteria from cancer patients with bacteremia (Table 3).

Microbes

Count   

VA

CN

CAZ

TZP

TE

AMC

CTX

CIP

RD

DO

C

PB

Pen

-

-

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

R

S

E. coli

16

40   

60   

62   

38   

30   

70   

18    

82   

53   

47   

53   

47   

47   

53   

23   

77   

51   

49   

32   

68   

54   

46   

53   

47   

81   

19   

Staph aureus

14

46

54

53

47

38

62

26

74

44

56

43

57

41

69

36

64

32

72

41

59

46

54

41

59

79

21

P. aeruginosa

14

 30

70

40

60

29

71

24

76

32

68

31

69

43

57

42

58

19

81

40

60

42

58

42

58

70

30

Salmonella spps

12

51

49

31

69

52

48

45

55

43

52

42

58

24

74

25

75

41

59

30

70

40

60

21

79

65

35

bacillus spp.

11

32

68

32

68

43

57

37

73

29

71

35

65

53

47

35

65

22

78

39

61

22

78

39

61

91

09

Enterobacter spps

10

21

79

12

88

34

56

28

72

46

56

44

56

62

38

53

47

41

59

37

63

32

68

43

57

75

25

Mycobacterium Spps

09

48

52

18

82

51

49

51

49

48

52

39

61

44

56

38

62

33

77

32

68

40

60

59

41

79

21

S. pyogene

09

30

70

36

64

41

59

39

61

45

55

27

73

53

47

43

57

19

81

38

62

20

80

61

39

59

41

H.pylori

08

46

54

27

73

52

48

44

56

28

72

45

55

32

67

50

50

22

78

36

64

42

58

46

56

76

24

Klebsiella spp.

07

40

60

45

55

35

65

12

88

23

77

26

74

61

39

29

71

40

60

40

60

61

39

49

51

62

38

S. epidermidis

05

25

75

33

77

24

76

29

71

38

62

48

52

41

59

24

76

29

71

57

43

45

55

48

52

45

55

Shigella

05

31

69

39

61

19

81

50

50

21

79

30

70

60

40

46

54

38

62

50

50

31

69

40

60

61

39

Table 3 Susceptibility profile of various isolated bacteria against various antibiotics

In the current study 100 blood samples were also screened for CRP. Out the of the total sample 50 blood samples taken from cancer patients and 50 from non-cancer patients. In total 50 cancer sample CRP analysed that 21 patents have the Lowest (0.92 mg/L), 08 was (0.92-1.93 mg/L) 11 was (1.94-3.69 mg/L) and 10 patients were on the Highest (>3.69 mg/L) risk. In 50 non-cancer blood sample the CRP level are 09 individual were (0.92 mg/L), 11 individual (0.92-1.93 mg/L), 25 individual (1.94-3.69 mg/L) and 03 patients were Highest (>3.69 mg/L) risk. Juan et al.23 also studied High levels (>3 mg/L) of CRP were associated with an increased risk of incident cancer (hazard ratio, 1.4; 95% CI, 1.1 to 1.7) compared with persons with low levels (<1 mg/L. Although CRP seems to affect several cancer sites, the association was strongest for lung cancer (hazard ratio, 2.8; 95% CI, 1.6 to 4.9). Decreased CRP levels was associated with an increased cancer risk of 2.6 (95% CI, 1.6 to 4.4) in homozygous carriers. The cancer and non-cancer samples were also screened for CBC analysis through Sysmex and cell dyne ruby. Durnaś et al.24 also reported the Calculation of blood sample permanence used for Complete Blood Count (CBC) by means of Sysmex XN-9000 also through Mindray BC-6800 analyzers. Similar screening method also showed by Daniluk et al.25 that the partiality for haematocrit, mean corpuscular haemoglobin attentiveness, mean corpuscular volume, and Red Blood Cell (RBC) sharing width coefficient of variant was greater than the critical dissimilarity after 8 hours. In the consequence the same analyzers were used (Figure 1 & 2).

Figure 1 CRP analysis for cancer samples.

Figure 2 CRP analysis for non-cancer samples.

Conclusions and recommendations

It is concluded that the maximum isolated bacteria were E. coli, S. aureus, P. aeruginosa and Salmonella. The ESBL producing bacteria were Enterobacter spp. E. coli, Klebsiella spp, P. aeruginosa, and Shigella. The blood analysis were performed for cancer patients in which 60 % patient were high level of WBC in cancer and 56% were High level of WBC in non-cancer patients. In urine samples the presence of bacteria in cancer which is 86% and 78% in non-cancer samples.

It is recommended that Continuous ESBL screening and supervision are necessary at hospital settings to observe and develop approaches for observing and controlling the spread of ESBL generating bacteria. Community awareness is required for use and mismanagement of antibiotics. Alternate approaches must be hopeful to find out novel sources of antibacterial for control of bacterial resistance. More research studies are required to study cancer and non- cancerous patients on microbiological, molecular and immunological level. Selective pressure for resistance through antimicrobial use is important, infection control practices are critical to limiting the spread of resistant organisms. The life-threatening nature of bacteremia and sepsis underscores the importance of using timely surveillance data to develop rational antimicrobial therapy recommendations and to design strategies to help control antimicrobial resistance.

Acknowledgments

None.

Conflicts of interest

Authors declare that there are no conflicts of interest.

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