1

Klebsiella pneumoniae

(

K. pneumoniae

) is a Gram-negative bacterium that can cause bacteremia, sepsis, urinary tract infections, pneumonia, and nosocomial infections ¹. A study conducted in hospitals in 10 Asian countries from 2008 to 2009 found

K. pneumoniae

as the most common cause of nosocomial infections, namely pneumonia associated with ventilator installation ². Antibiotics resistance has become a significant problem worldwide. In the era of increasing antibiotic resistance and the lack of discovery of new antibiotics, it is necessary to optimize the use of existing antibiotics to treat infections ³. Antibiotic resistance of

Klebsiella

spp. is highest in Asia (≥60%), reflecting an alarming increase in opposition to this bacterium. Increased resistance, especially to various classes of antibiotics classified by WHO as essential drugs, so the used of new and more broad-spectrum antibiotics must be limited if there are still narrower-spectrum and effective antibiotics ⁴.

The prevalence of

K. pneumoniae

infection was 13% in America, 5% in Pakistan, 64.2% in Nigeria, 33.9% in India, 17.4% in Denmark, and 14.1% in Singapore.

K. pneumoniae

Extended-Spectrum Beta-Lactamase (ESBL) infection in Indonesia was 35.35%, a total of 297 isolates from patients hospitalized from January–April 2005 and 38.5% of all isolates from October 2014–May 2015 at Surabaya ^5,6. In a study conducted in Turkey in 2016, there were 190 patients with nosocomial bacteremia caused by

K. pneumoniae

with a mortality rate of 47.9% ⁷. Another study in Taiwan in 2020 involving 150 patients with bacteremia caused by

K. pneumoniae

had a mortality rate of 20–40% ⁸.

Ciprofloxacin, cotrimoxazole, and doxycycline are antibiotics used for a long time and are included in WHO essential drugs because of their excellent efficacy, minimal side effects, and relatively inexpensive. There are also injection and oral dosage forms with good bioavailability making them easier to use and relatively easy to obtain. The 2021 hospital antimicrobial stewardship guidelines from the Ministry of Health also classify these antibiotics in the access group ^9,10. These antibiotics are still effective enough to treat various Gram-negative infections based on available data, including

K. pneumoniae

. The study in France with 40 samples of

K. pneumoniae

, 75% susceptible to ciprofloxacin, also analyzed the time-kill curve of ciprofloxacin against

K. pneumoniae

¹¹. A study in China demonstrated the use of cotrimoxazole for

K. pneumoniae

in vitro. Of 812 isolates of

K. pneumoniae

, 175 (21.6%) isolates were resistant to cotrimoxazole ¹². A study in Mumbai, India, with a retrospective method involving 2951 samples collected from January 2017–December 2018 with 263 of these samples were isolates of

K. pneumoniae

from sputum, showed doxycycline susceptibility of 68.4% ¹³. Clinical infectious disease stated that based on pharmacokinetic and MIC data for UTIs caused by

Enterobacter

ESBL, doxycycline could potentially be used for therapy ¹⁴.

Time kill curve can be used to study antimicrobial activity that depends on concentration and time-dependent, so this method serves as an alternative option that provides more detailed and dynamic information than MIC. Considering the sensitive profile data to ciprofloxacin, cotrimoxazole, and doxycycline antibiotics, this study aimed to compare the effectiveness of these antibiotics in vitro against

K. pneumoniae

and

K. pneumoniae

ESBL isolates at a hospital in the form of an analysis of the bacterial time-kill curve.

2

The study used a case-control (experimental) analysis with a posttest control group design. The subjects used were

K. pneumoniae

ESBL and non-ESBL, replicated 6 times. Antibiotics used included ciprofloxacin, cotrimoxazole, and doxycycline. This study was conducted from June 2021 to May 2022. The antibiotic doses used in

K. pneumoniae

varied, including ciprofloxacin 0.25, 0.5, 1 p/mL, cotrimoxazole 2, 4, and 8 p/mL, and 4, 8, and 16 p/mL. Furthermore, the time-kill was evaluated for each antibiotic exposure, including 0, 2, 4, 6, 8, and 24 h.

Before the examination, the number of colonies in the CFU/mL log was calculated first. If clinical isolates were found stored with the identification of fungi, sterile culture results, and clinical isolates of

K. pneumoniae

non-ESBL and

K. pneumoniae

ESBL were identified and tested for antibiotic susceptibility. Manually, the isolate could not be used. The measurement results were analyzed using statistical product and service solution (SPSS) software version 26.0 (IBM Corp., Armonk, NY, USA). The analysis used were ANOVA, post-Hoc, Mann Whitney, and Kruskal Willis with

p

< 0.05.

3

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL,

and K. pneumoniae

ATCC on administering of ciprofloxacin, doxycycline, and cotrimoxazole 1 MIC at 2–8 h all showed bacteriostatic activity with a reduction in colony number of 1-2-log CFU/mL.

K. pneumoniae

non-ESBL,

K. pneumoniae

ATCC, administration of ciprofloxacin, doxycycline 2 MIC at 2–6 h showed bacteriostatic activity with a reduction in colony number of 1-2-log CFU/mL while at 8 h showed bactericidal activity with a reduction in the number of colonies by 3 log CFU/mL. Administration of cotrimoxazole 2 MIC at 2–8 h showed bacteriostatic activity with a reduction in the number of colonies by 1-2-log CFU/mL.

K. pneumoniae

ESBL on the administration of ciprofloxacin, doxycycline, and cotrimoxazole 2 MICs at 2–8 h showed bacteriostatic activity with a reduction in colony number of 1-2-log CFU/ml. The distribution of time-kill

K. pneumoniae

after antibiotic administration could be seen in

Figs. 1–6

.

Time-kill graph of the number of non-ESBL

K. pneumoniae

colonies at 0, 2, 4, 6, 8, and 24 h against antibiotics ciprofloxacin, doxycycline, and cotrimoxazole at 1 MIC, 2 MIC, 4 MIC.

Time-kill graph of the number of

K. pneumoniae

ESBL colonies at 0, 2, 4, 6, 8, and 24 h against antibiotics ciprofloxacin, doxycycline, and cotrimoxazole at 1 MIC, 2 MIC, 4 MIC.

Time-kill graph of the number of

K. pneumoniae

ATCC colonies at 0, 2, 4, 6, 8, and 24 h against the antibiotic’s ciprofloxacin, doxycycline, and cotrimoxazole at 1 MIC, 2 MIC, 4 MIC.

Time-kill graph of the number of colonies of

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL, and

K. pneumoniae

ATCC at 0, 2, 4, 6, 8, and 24 h against the administration of the antibiotic ciprofloxacin at concentrations of 1 MIC, 2 MIC, 4 MIC.

Time-kill graph of the number of colonies of

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL, and

K. pneumoniae

ATCC at 0, 2, 4, 6, 8, and 24 h against doxycycline antibiotics at concentrations of 1 MIC, 2 MIC, 4 MIC.

Time-kill graph of the number of colonies of

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL, and

K. pneumoniae

ATCC at 0, 2, 4, 6, 8, and 24 h against cotrimoxazole antibiotics at concentrations of 1 MIC, 2 MIC, 4 MIC.

K. pneumoniae

non-ESBL,

K. pneumoniae

ATCC on the administration of ciprofloxacin 4 MIC at 2–4 h showed bacteriostatic activity with a reduction in colony number of 1-2-log CFU/mL while at 6–8 h showed activity bactericidal with a reduction in the number of colonies by 3-log CFU/mL. In

K. pneumoniae

ESBL on the administration of ciprofloxacin 4 MIC at 2–6 h showed bacteriostatic activity with a reduction in the number of colonies by 1-2-log CFU/mL, while at 8 h showed bactericidal activity with a reduction in colony number by 3-log CFU/mL.

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL, and

K. pneumoniae

ATCC on the administration of doxycycline 4 MIC at 2–6 h showed bacteriostatic activity with a reduction in the number of colonies by 1-2-log CFU/mL while at 8 h showed activity bactericidal with a reduction in the number of colonies by 3 log CFU/mL. Administration of cotrimoxazole 4 MIC at 2–8 h showed bacteriostatic activity with a reduction in the number of colonies by 1-2-log CFU/mL.

K. pneumoniae

non-ESBL,

K. pneumoniae

ESBL,

K. pneumoniae

ATCC, on the administration of ciprofloxacin, doxycycline, cotrimoxazole 1 MIC, 2 MIC, 4 MIC showed that all colonies began to grow back at 24 h. Results of the analysis of the effectiveness of time-kill

K. pneumoniae

could be seen in

Tables 1 and 2

.

Table 1

Analysis of differences in the effectiveness of antibiotics used in

K. pneumoniae

.

Time to Kill	Antibiotic		CI 95%	p -value
2	Ciprofloxacin	Doxycycline	0.023–0.258	0.020
	Doxycycline	Cotrimoxazole	−0.161– 0.074	0.453
	Cotrimoxazole	Ciprofloxacin	0.067–0.302	0.003 a
4	Ciprofloxacin	Doxycycline	0.089–0.407	0.004 a
	Doxycycline	Cotrimoxazole	0.038–0.356	0.017
	Cotrimoxazole	Ciprofloxacin	0.286–0.603	<0.001 a
6	Ciprofloxacin	Doxycycline	−0.004– 0.451	0.054
	Doxycycline	Cotrimoxazole	−0.027– 0.427	0.082
	Cotrimoxazole	Ciprofloxacin	0.196–0.651	0.001 a
8	Ciprofloxacin	Doxycycline	–	0.015
	Doxycycline	Cotrimoxazole	–	0.015
	Cotrimoxazole	Ciprofloxacin	–	0.005 a
24	Ciprofloxacin	Doxycycline	0.241–0.818	0.001 a
	Doxycycline	Cotrimoxazole	−0.075– 0.502	0.140
	Cotrimoxazole	Ciprofloxacin	0.454–1.032	<0.001 a

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