Assessment of the activity of tigecycline and colistin on biofilm producer Klebsiella pneumoniae isolated from COVID-19 patients, Iran

doi:10.21203/rs.3.rs-2196378/v1

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Assessment of the activity of tigecycline and colistin on biofilm producer Klebsiella pneumoniae isolated from COVID-19 patients, Iran

https://doi.org/10.21203/rs.3.rs-2196378/v1

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Background

Pneumonia caused by β-lactamase-producing Klebsiella pneumoniae is encountered in hospitals. We aimed to investigate the activity of tigecycline, colistin and other selected antibiotics against 27 β-lactamase-producing Klebsiella pneumonia isolated from bronchoalveolar lavage (BAL) in COVID-19 patients.

Methods

In the current survey phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. Biofilm formation was assessed by staining with 1% crystal violet. Finally, a set of the virulence-encoding genes were detected by the PCR method.

Results

This study demonstrated the high percentage of ESBL and carbapenemase-producing Klebsiella pneumoniae among COVID-19 patients. Tigecycline and colistin were more effective against these isolates. ESBL associated genes were detected in (18 (66.7%) bla_TEM, 27 (100%) bla_SHV, and 18 (66.7%) bla_CTX−M) isolates. Also, carbapenemase-related genes were detected in 16 (59.3%) isolates. The prevalence of these genes were bla_{OXA−48−like} 14(51.9%), and bla_NDM1 2 (7.4%). Twenty-seven isolates exhibited biofilm formation. Molecular distribution of virulence genes revealed that 92.59%, 92.5%, 88.88%, 11.11% and 18.5% of the isolates carried entB, mrkD, Irp2, fimH and magA genes, respectively. MLST results for four colistin-resistant isolates showed three different sequence types-ST: ST3500, ST273, and 2 cases of ST2558.

Conclusion

The results of this study demonstrated the prevalence of infections caused by β-lactamase-producing Klebsiella pneumoniae, which are biofilm producers among respiratory hospitalized Patients. The effective antimicrobial activity of tigecycline to the bacteria that produce these enzymes may be efficient in faster and better treating COVID-19 patients which are hospitalized.

Tigecycline

Klebsiella pneumoniae

COVID-19

The prevalence of bacterial co-infection with coronavirus disease (COVID-19) has been different, but it may be as high as 50% in non-survivors (1). In Wuhan, 15% of hospitalized Patients with COVID-19 were reported to have bacterial co-infection (2). According to current findings, the top bacteria of secondary bacterial infections, which were detected in COVID-19 patients, were Mycoplasma pneumonia, Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii, and stenotrophomonas maltophila. These findings support the routine use of antibiotics in the management of treatment of infection associated with COVID-19. COVID-19 patients are hospitalized for a long time under invasive mechanical ventilation, which makes them more exposed to nosocomial infections (3). National Institute for Health and Care Excellence (NICE) COVID-19 recommended antibacterial treatment for patients with suspected bacterial infections, patients with unknown sources of infection and worrying symptoms, or high-risk patients with untreated bacterial infections (1). Nosocomial acquired Klebsiella infections are wide spread resulting in high morbidity and mortality because of limited number of antibiotics treatment options. Therefore, for the treatment of infections caused by extended-spectrum-β-lactamase (ESBL) producing Klebsiella pneumoniae, carbapenems have been considered suitable options for infection control. However, the overuse of carbapenems in hospitals has led to an increase in Carbapenem-Resistant Klebsiella pneumoniae (4). Klebsiella pneumoniae carbapenemase (KPC)-producing is becoming distressing (5). Several mechanisms result in resistance to carbapenems, including the production of carbapenemases of class A (KPC, GES, and others), class B (mainly IMP, VIM, or NDM), and class D (OXA-48) and related enzymes. For the carbapenem-resistant isolates infections treatment, tigecycline which is one of the glycylcycline derivatives of minocycline is considered the last resort (6). Bacterial attachment and biofilm formation via surface attachment structures such as adhesions and capsular polysaccharides have led to defeat in infection removal (7). In the current study, we undertook a retrospective observational analysis of patients with confirmed SARS-CoV-2. The purpose of this study was to evaluate the effect of tigecycline and other selected antibiotics against COVID-19 co-infections related to ESBL /carbapenemase-producing K. pneumoniae. This study determines the prevalence of gene profile of ESBL, AmpC, carbapenemase, and co-harbored virulence-associated genes in K. pneumoniae isolated from COVID-19 patients in IRAN.

Bacterial strains

Twenty-seven β-lactamase-producing K. pneumoniae strains from 100 respiratory tract samples (bronchoalveolar lavage (BAL)) of severe COVID-19 patients with bacterial coinfections were collected from October 2020 to February 2021. The isolates were identified, using standard phenotypic microbiological tests and API 20E commercial strips (bioMérieux, France). All isolates confirmed to be K. pneumoniae using 16S rRNA sequence analysis after PCR amplification with the universal primers (27F: AGAGTTTGATCCTGGCTCAG and 1429R: GGTTACCTTGTTACGACTT) (8). Isolates were stored in − 20°C in TSB containing 20% glycerol until further studies.

Antibiotic Susceptibility Testing

Antimicrobial susceptibility test was performed by the disk diffusion methods according to the Clinical and Laboratory Standards Institute (CLSI; 2021) (9). Antibiotic disks include: levofloxacin (LEV) (5 µg), azithromycin (AZ) (15 µg), ceftazidime (CAZ) (30 µg), amikacin (AN) (30 µg), gentamicin (GN) (10 µg), cefepime (FEP) (30 µg), imipenem (IMP) (5 µg), meropenem (MEN) (5 µg), piperacillin-tazobactam (PTZ) (100/10 µg), piperacillin (PIP), ciprofloxacin (CP) (5 µg), cotrimoxazole (SXT) (25µg), cefotaxime (CTX) (30 µg), tobramycin (TOB) (10µg), ceftazidime/Clavulanic Acid (CAZ) and cefoxitin (FOX) (10µg) (9). The MICs of colistin and tigecycline were determined using the broth microdilution method and the results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoint recommendations (10). Escherichia coli ATCC 25922, and K. pneumoniae ATCC 700603 were included in each run as control. The XDR, MDR, and non-MDR according to the international expert proposal for interim standards guidelines (11), as follows: XDR was defined as acquired resistance to ≥ 1 agent in all but ≤ 2 categories, MDR as resistance to ≥ 1 agent in ≥ 3 antimicrobial categories and, non-MDR as resistance to 0–2 antimicrobial categories.

Phenotypic Screening of ESBL, AmpC betalactamase, and Carbapenemase Producer-K. pneumonia

Based on guidelines of the Clinical Laboratory Standards Institute (CLSI 2021) combined disk method was used for screening ESBL production among K. pneumonia. Briefly, susceptibility to cefotaxime (30 µg), cefotaxime/clavulanate (30/10 µg), ceftazidime (30 µg), and ceftazidime/clavulanate (30/10 µg) (Mast Co., UK) was determined on Muller-Hinton agar (Merck Co, Germany). ESBL-producing test result was defined as an increase in the diameter of the area around the ceftazidime/clavulanate and cefotaxime/clavulanate disks by ≥ 5 mm compared to the discs without clavulanic acid (9). E. coli ATCC 35218 was used as control strain. Cefoxitin disk (30 µg) was used to screen AmpC-producing isolates. A double-disk synergy test was performed with cefoxitin-EDTA to determine AmpC production (12). To screen carbapenemase-producing isolates the modified Hodge test (MHT) was performed. K. pneumoniae ATCC BAA- 1705 and BAA-1706 were used as MHT-positive and -negative controls (9).

Detection Of Esbl, Ampc, And Carbapenemase-related Genes

The polymerase chain reaction was performed with primers to detect genes encoding AmpC (bla_ACC, bla_DHA, bla_EBC, bla_FOX, bla_MOX, and bla_CIT), ESBLs (bla_TEM, bla_SHV, and bla_CTX−M), and carbapenemase (bla_IMP, bla_VIM, bla_NDM1, bla_KPC, and bla_{OXA−48−like}) (13, 14). All primer sequences used are listed in Table 1. The products were separated by electrophoresis in 1% agarose gel with 1× TBE (Tris/ borate/EDTA) buffer, stained with safe stain load dye (CinnaGen Co., Tehran, Iran), and visualized under ultraviolet illumination.

Table 1

**Primers** used for identification of *Klebsiella pneumoniae* genes for encoding AmpC, ESBLs and carbapenemase.
(bp) Size	Sequence (5' to 3')	Primer	Target	References
383	CGTCTAGTTCTGCTGTCTTG	F	KPC	In study
383	GCGGCGTTATCACTGTATTG	R	KPC	In study
487	GGCGTAGTTGTGCTCTGG	F	OXA-48	In study
487	TATAGTCACCATTGGCTTCGG	R	OXA-48	In study
232	ATCCACTATCGCCAGCAG	F	SHV	In study
232	CCTCATTCAGTTCCGTTTCC	R	SHV	In study
552	AGGAAGTGTGCCGCTGTATG	F	CTX-M	In study
552	CTGTCGCCCAATGCTTTACC	R	CTX-M	In study
373	TCGCCGCATACACTATTCTC	F	TEM-1	In study
373	AACTTTATCCGCCTCCATCC	R	TEM-1	In study
395	ATACCGCCTGGACCGATGAC	F	NDM-1	In study
395	GAGATTGCCGAGCGACTTGG	R	NDM-1	In study
480	TGTCGCAAGTCCGTTAGC	F	VIM	In study
480	GCAGCACCAGGATAGAAGAG	R	VIM	In study
335	TTAGCGGAGTTAGTTATTGGC	F	IMP	In study
335	TTAGTTACTTGGCTGTGATGG	R	IMP	In study
520	GCT GCT CAA GGA GCA CAG GAT	F	MOX	(42)
520	CAC ATT GAC ATA GGT GTG GTG C	R	MOX	(42)
190	AAC ATG GGG TAT CAG GGA GAT G	F	FOX	(42)
190	CAA AGC GCG TAA CCG GAT TGG	R	FOX	(42)
462	TGG CCA GAA CTG ACA GGC AAA	F	CIT	(42)
462	TTT CTC CTG AAC GTG GCT GGC	R	CIT	(42)
405	AAC TTT CAC AGG TGT GCT GGG T	F	DHA	(42)
405	CCG TAC GCA TAC TGG CTT TGC	R	DHA	(42)
346	AAC AGC CTC AGC AGC CGG TTA	F	ACC	(42)
346	TTC GCC GCA ATC ATC CCT AGC	R	ACC	(42)
302	TCG GTA AAG CCG ATG TTG CGG	F	EBC	(42)
302	CTT CCA CTG CGG CTG CCA GTT	R	EBC	(42)

Detection of mcr-1-5 genes

The PCR testing was conducted for plasmid-mediated colistin resistance detection associated with mcr-1-5 (15).

Multilocus Sequence Typing (Mlst)

Strain typing among four colistin-resistant K. pneumoniae isolates was examined by MLST. (16)was performed on all four isolates following the protocol described on the Pasteur MLST site (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html)(17). All primer sequences used in MLST are listed in Table 2.

Table 2

Primers used for identification of Strain Typing (MLST) of *Klebsiella pneumoniae* (43)
Gene name	Primer name	Sequences (5’ to 3’ end)	Amplicon size
gapA	gapA-F	TGAAATATGACTCCACTCACGG	662
	gapA-R	CTTCAGAAGCGGCTTTGATGGCTT
infB	infB-F	CTCGCTGCTGGACTATATTCG	462
	infB-R	CGCTTTCAGCTCAAGAACTTC
mdh	Mdh-F	CCCAACTCGCTTCAGGTTCAG	756
	Mdh-R	CCGTTTTTCCCCAGCAGCAG
pgi	Pgi-F	GAGAAAAACCTGCCTGTACTGCTGGC	718
	Pgi-R	CGCGCCACGCTTTATAGCGGTTAAT
phoE	PhoE-F	ACCTACCGCAACACCGACTTCTTCGG	602
	PhoE-R	TGATCAGAACTGGTAGGTGAT
rpoB	RpoB-F	GGCGAAATGGCWGAGAACCA	1075
	RpoB-R	GAGTCTTCGAAGTTGTAACC
wzi	Wzi-F	GTGCCGCGAGCGCTTTCTATCTTGGTATTCC	580
	Wzi-R	GAGAGCCACTGGTTCCAGAAYTTSACCGC

Biofilm Formation Assays

The biofilm formation capacity of all strains were determined by crystal violet staining method described previously (26). Briefly, Biofilm formation was conducted by growing bacteria isolates in a flat bottom (96 well). A 200-µL 0.5 McFarland suspension was incubated in each well of a 96-well plate at 37°C for 48h. After, the plates were washed three times with PBS, and each well was stained with 200 µL of 1% crystal violet for 20 min at ambient temperature. The plates were again washed three times to remove excess stain. The crystal violet attached to the adherent bacteria was solubilized with 180 µl of 33% glacial acetic acid and the absorbance was read at OD570. Un-inoculated LB medium was used as a negative control, while the reference strain ATCC 700603 was selected as positive control. (18).

Detection Of Virulence Genes

The k. pneumoniae isolates were screened by PCR for the following virulence genes: the type 1 adhesion (fimH), type-3 adhesion (mrkD), enterobactin (entB), yersiniabactin high-pathogenicity island (YHPI) (irp-2), capsular polysaccharide (magA, wcaG) and type 3 fimbriae (MrkA) (19, 20). The primers used to identify these genes were designed using Allele ID 6 software and BLAST using the program on the NCBI website. All primer sequences used are listed in Table 3.

Table 3

Primers used for identification of virulence genes of *Klebsiella pneumoniae*
Target gene	Primer sequence (5´→3´)	Amplicon size	Annealing temperature	Reference
fimH	F: GCTGCTGCTGGGCTGGTC R: GGTCGGGAACGGGTAAGAGG	292 bp	65˚ C	In study
mrkA	F: AATGTAGGCGGCGGTCAG R: CTCTCCACCGATAACGCCA	351 bp	59˚ C	In study
mrkD	F: CTGAGTGAAACGGGATATGC R: AGCGGTATGGTGATGTAGC	224 bp	58˚ C	In study
magA	F: CATTGCCGCTACTACAGGAG R: AGTGAACGAATTGATGCTTGG	239 bp	60˚ C	In study
entB	F: GCATCGGTGGCGGTGGTC R: CGGCGAACAAGGTCAACTGG	439 bp	63˚ C	In study
Irp2	F: GCAACGGCGGGCATAGTC R: GCGAGGTCTGGCTACAATGG	320 bp	63˚ C	In study
wcaG	F: AGCAACCGATTAGTGAGTCC R: TCAACGCCAGTGCCTACG	402 bp	58˚ C	In study

Statistical Analysis

The statistical analysis was performed by using SPSS version 22 (SPSS Statistics, Inc., Chicago, IL, USA). In all statistical tests, P < 0.05 was considered significant.

Antimicrobial Susceptibility

Based on susceptibility testing results, 16 (59.3%) and 11 (40.7%) isolated strains were categorized as Multidrug-resistant (MDR) and Extensively-drug resistant (XDR) strain respectively (Table 4). In this study, we determined the antimicrobial susceptibility patterns by micro-broth dilution method for tigecycline and colistin. The MICs range of ESBL/CRKP isolates against tigecycline and colistin was 0.25–0.5 and 2–16 mg/L, respectively. Tigecycline was sensitive against all ESBL/CRKP isolates. The highest resistance rate in this study was against azithromycin (100%), and ceftazidime (85.18%) followed by cefotaxime (92.5%) (Fig. 1). Through the broth microdilution test it is revealed that tigecycline and colistin were highly sensitive (100% and 85.2%) (Table 4). Another antibiotic with higher sensitivity was amikacin (44.4%). Phenotypic ESBL detection tests indicated that 27 (100%) K. pneumoniae isolates were ESBL producers, and they were all sensitive to tigecycline.

Table 4

Antibiotic resistance profiles and MICs of tigecycline and colistin of twenty-seven ESBL /CRKP *Klebsiella pneumoniae* isolates.
Isolates	ESBL genotype	Carbapenemase genotype	MIC ( mg/L ) Colistin	MIC (mg/L) Tigecycline	MDR/XDR	Antibiotic resistance profile	Biofilm
KP1	SHV, CTX-M, TEM	OXA-48	2	0.5	MDR	PTZ,LEV,FEP,IPM,MEN,PTZ,CAZ,CZA,AZ, PIP,CTX,FOX	Intermediate
KP2	SHV, CTX-M, TEM	-	0.5	0.5	MDR	FEP, PTZ, CRO,SXT,AZ, LEV,PIP,CTX	Intermediate
KP3	SHV	-	0.5	0.25	MDR	LEV,AZ,CZA, CAZ, SXT,PIP,CTX	strong
KP4	SHV, CTX-M, TEM	-	16	0.5	XDR	CZA,AN,GM,TOB,CTX, FEP ,MEN, AZ,PTZ,CAZ,CP, CRO, SXT,CL	Strong
KP5	SHV, CTX-M, TEM	OXA-48	0.5	0.5	XDR	LEV,AZ,CZA, AN,GM,FEP,TOB IPM,MEN,PTZ,CRO,SXT,CAZ,CP,CTX,FOX	Strong
KP6	SHV ,CTX-M, TEM	-	16	0.25	MDR	CL,LEV,CZA ,AZ,CTX,FOX	Strong
KP7	SHV,TEM	-	0.5	0.5	MDR	CTX,AZ,CZA, FEP,IPM,MEN, PTZ,CAZ,CP, CRO	Intermediate
KP8	SHV, CTX-M, TEM	-	0.5	0.5	MDR	CTX,AZ,CZA, FEP,IPM,MEN ,PTZ,CAZ, CP, SXT	Strong
KP9	SHV	OXA-48	0.5	0.25	MDR	CZA,FEP,IPM,MEN PTZ,CAZ,CP, SXT,PIP	Intermediate
KP10	SHV, CTX-M, TEM	-	0.5	0.5	MDR	AZ,CAZ,PIP, CRO,SXT, CTX	weak
KP11	SHV, TEM	OXA-48	8	0.5	XDR	CL,CZA,AN, GM,FEP ,CTX, MEN,PTZ,CAZ, CP, SXT,FOX, TOB,LEV,AZ,PIP	Strong
KP12	SHV, CTX-M, TEM	OXA-48	0.5	0.25	MDR	AZ, FEP,IPM,MEN,PIP, PTZ,CTX,GM, CAZ,CP	weak
KP13	SHV, CTX-M, TEM	OXA-48	0.5	0.5	XDR	LEV,AZ,CZA, AN,GM,FEP,TOB IPM,MEN,PTZ,CRO,SXT,CAZ,CP,CTX,FOX	Strong
KP14	SHV, CTX-M, TEM	NDM-1	0.5	0.25	MDR	CTX, FEP, CAZ,FOX	Intermediate
KP15	SHV, CTX-M	OXA-48	0.5	0.5	XDR	LEV,AZ,CZA,AN,FOX FEP,IPM,GM,CTX MEN,PTZ,CAZ, CP,CRO,SXT	Strong
KP16	SHV	-	0.5	0.5	MDR	AZ,CZA,AN, FEP,IPM,MEN, PTZ,CAZ,CP, CTX,SXT	weak
KP17	SHV, TEM, CTX-M	OXA-48	0.5	0.25	XDR	LEV,AZ,CZA,AN,GM,FEP,IPM,MEN,PTZ,CAZ,CTX, GM,FEP,IPM, MEN,PTZ,CAZ, CP,CRO,SXT	strong
KP18	SHV, CTX-M	OXA-48	2	0.5	MDR	CTX,AZ,CZA, GM,FEP,IPM, MEN,PTZ,CAZ,	weak
KP19	SHV, TEM	NDM-1	0.5	0.5	MDR	CZA,AN,GM, FEP,LEV,IPM, AZ,PTZ,CAZ,CP, SXT,FOX	Intermediate
KP20	SHV, CTX-M, TEM	OXA-48	0.5	0.25	MDR	LEV,AZ,FEP, CZA, IPM,MEN, PTZ, CAZ, CP,CTX,SXT	weak
KP21	SHV, CTX-M	OXA-48	8	0.25	XDR	CL,LEV,AZ,AN,CZA,FEP,IPM,CP,CRO,SXT, PTZ,CAZ,FOX,TOB,CTX	Intermediate
KP22	SHV, CTX-M, TEM	OXA-48	1	0.5	XDR	LEV,AZ,CZA,AN,GM,FEP,IPM,CTX MEN,PTZ,CAZ, CP,CRO,SXT,FOX	Intermediate
KP23	SHV, CTX-M	OXA-48	2	0.5	XDR	LEV,AZ,CZA, GM,FEP,IPM, MEN,CP,SXT, CRO,FOX,CAZ, CTX	strong
KP24	SHV, CTX-M, TEM	-	0.5	0.5	MDR	AZ,CRO,PTZ,CTX	weak
KP25	SHV, CTX-M, TEM	OXA-48	2	0.5	XDR	LEV,AZ,CZA, FEP,IPM,MEN, PTZ,CAZ,CP, CRO,SXT,AM,FOX, GM,PIP,CTX	strong
KP26	SHV, CTX-M, TEM	-	2	0.5	XDR	LEV,AZ,CZA,AN,GM,FEP,IPM,CTX, MEN,PTZ,CAZ, CP,SXT,CRO,PIP ,FOX	Strong
KP27	SHV	-	1	0.5	MDR	AZ,CZA,SXT,FOX,AN,CTX	Intermediate

Genotypic Assays For Esbl, Ampc, And Carbapenemase

None of the strain harbored AmpC-associated genes (bla_ACC, bla_DHA, bla_EBC, bla_FOX, bla_MOX, and bla_CIT). Modified Hodge test phenotypicaly indicated that 21 (77.7%) K. pneumoniae isolates were carbapenemase producers.

Molecular methods revealed that 16 (59.3%) K. pneumoniae isolates harbored carbapenemase-related genes. The prevalence rates of these genes were bla_{OXA−48−like} 14(51.9%), and bla_NDM1 2(7.4%). None of the strain harbored bla_IMP, bla_VIM and bla_KPC genes. Twenty- seven (100%) K. pneumoniae isolates were positive for the at least one of the ESBL-related genes (Fig. 2) and (Fig. 3). Distribution of these genes among K. pneumonia strains were 18 (66.7%) bla_TEM, 27 (100%) bla_SHV, and 18 (66.7%) bla_CTX−M (Table 4). Mcr-1-5 genes were not detected in K. pneumoniae isolates in the current study.

Molecular Typing

MLST analysis of four colistin-resistant K. pneumoniae revealed different STs and their STs were as follows: ST3500, ST273, and 2 cases of ST2558.

Assessment Of Biofilm Formation Capacity

All K. pneumoniae isolates were determined to develop biofilm, 12 (44.44%) formed fully established biofilms, 9 (33.33%) were categorized as moderately biofilm-producing, 6 (22.22%) formed weak biofilms.

Assessment Of Virulence Factors

In general, five of the 7 screened virulence factors (fimH, irp2, mrkD,magA, and entB) were identified in the 27 K. pneumonia isolates. All K. pneumoniae isolates carried at least one biofilm related genes. Molecular distribution of virulence genes revealed that 25 (92.59%) biofilm producer isolates harbored entB and mrkD virulence genes and 24 (88.88%), 3(11.11%), and 5 (18.5%) of these isolates harbored Irp2, fimH and magA virulence genes. The number of positive virulence genes determinants varied from two to five genes in any isolate. The genes of wcaG and mrkA were not detected in any k. pneumonia isolate. Different percentages of fimbriae genes were identified, while 11.1% of fimH gene was detected, for mrk A gene none of isolates were positive.

The Correlation Between Biofilm Formation And Antibiotic Resistance Phenotypes

The majority of strong biofilm-forming K. pneumoniae isolates were XDR. Only 25% of MDR isolates were strong biofilm producers, whereas 73% of XDR isolates form strong biofilms (Fig. 4). It should be noted that all isolates which were positive in fimH and magA genes were belong to XDR strains.

Association between the presence of virulence gene and biofilm formation

According to PCR results for detecting virulence genes, the presence of the fimH gene was confirmed among three strong biofilm-producers. Moreover, four of the strong biofilm-producers had magA gene, while one of the intermediate biofilm-producers was positive for the presence of this gene. The entB and mrkD virulence genes were positive in the most of isolates, except in one weak biofilm isolates and one moderate biofilm isolate. The presence of the irp2 gene was confirmed among strong, moderate biofilm-producers, and 3 (50%) weak biofilm-producers.

The aim of the current study was to provide a point of reflection about the risk of ESBL/CRKP colonization and hospital-acquired infection in COVID-19 patients. Among the isolates of K. pneumoniae, a majority of them were producers of ESBL and Carbapenem-resistant K. pneumoniae (CRKP). In this study, 50% and 56.2% of isolates were resistant to meropenem and imipenem, respectively. Surprisingly, tigecycline was the most effective antimicrobial agent against these isolates. Other drugs in our study with higher sensitivities were colistin (85.2%) and amikacin (44.4%) respectively. The results of this study are consistent with the results of previous studies, which investigated the sensitivity of tigecycline (88.6% susceptibility) and colistin (73.9%) to carbapenem-resistant Enterobacterales (21, 22). It indicates a high sensitivity rate to tigecycline, which is still the best choice for MDR-CRE isolates.

Based on the recent reports, it was found that tigecycline is one of the most active antimicrobial agents against gram-negative and gram-positive isolates including drug-resistant pathogens (23). Among 27 K. pneumoniae isolates, 14 (51.8%) isolates were positive for the bla_OXA−48–type gene, which did not demonstrate the co-existence of other carbapenemases except for bla_NDM−1. This is reflective of high prevalence of OXA-48-positive K. pneumoniae in this study. NDM-1 was the second most frequent carbapenemase by 2 (7.4%) isolates. Similarly, bla_OXA−48 had first been identified in K. pneumoniae in Turkey, and it has recently been reported in the Middle East (24). OXA-48 is considered to be the most common carbapenemase in Middle-Eastern countries (24). The bla_NDM1 gene was reported first in India and was recently reported in Europe, North America, Asia, and Australia (24). Simultaneity of bla_NDM and bla_OXA−48 genes among K. pneumoniae have also been identified in several countries (25). The high prevalence of bla_OXA−48 and bla_NDM1 genotypes may be explained by the fact that Iran takes a large number of immigrants or visitors from bla_OXA−48 and bla_NDM high prevalence countries. Moreover, this study also revealed that 3 types of enzymes (VIM, IPM, and KPC) were not the significant types of carbapenemases. Consistent with the results of the study conducted by Gheitani et al., the results of this study showed that the prevalence rates of bla_VIM, bla_IMP, and bla_KPC were 4 (2.18%), 1 (0.5%), and 0%, respectively (26).

The distribution of bla_SHV, bla_CTX−M, and bla_TEM genes in ESBL strains were 27(100%), 18(66.7%), and 18(66.7%), respectively. These results showed a high proportion of SHV, CTX-M, and TEM enzymes among ESBL-producing K. pneumoniae strains in Covid-19 hospitalized patients. Our data were consistent with other studies conducted in Iran (27, 28). It seems that isolates harbored bla_SHV is the predominant genotype in this study. There are reports presenting bla_SHV as the most prevalent gene in many parts of the world (29). The situation related to ESBL production in Iran is very different, ranging from 9.8–75.7% (30–32). According to reports, ESBL production in Taiwan, Thailand, India, Pakistan, and South Korea are 8–29%, 64%, 44%, 36–56%, and 22%, respectively (31, 33). In this study, no genes related to blaAmpC were detected. On the other hand, perhaps the PCR results in our study were inconsistent with some surveys conducted in other parts of the world, due to genetic differences in causative strains, the use of antibiotics, and access to broad-spectrum and new antibiotics (34). The MDR/XDR isolates harbored ESBL/CRKP genes render most antibiotic mono-therapies ineffective. In accordance with the susceptibility results of our study, tigecycline, colistin, and amikacin have effective in vitro activity against ESBL/CRKP. Another study conducted in 18 European countries, indicated that the susceptibility rate of tigecycline to carbapenem resistance Enterobacteriaceae is 88.6%, which is in line with our findings (21). Tigecycline, colistin and some aminoglycosides still show favorably in vitro activities against carbapenem-resistant Enterobacteriaceae (35).

In the USA it was reported that 9.2% (MIC 8 mg/L, FDA) of MDR K. pneumoniae are resistance to tigecycline (36), while in Spain the rate of ESBL-producing isolates was 33.3% (MIC > 2 mg/L, EUCAST) (36). In conclusion, it can be suggested that against CRKP isolates harboring mcr genes use of combinatorial pharmacodynamics of colistin and tigecycline is more effective. Also, for preventing the increased resistance to colistin it has better suppression ability, and ability for decreasing colistin and tigecycline MICs (37). Increasing antibiotic resistance among biofilm-producing isolates raises serious concerns about limited treatment options in hospitals. Based on the surveys, substantial actions and introduction of new strategies are needed to control K. pneumoniae biofilm-related infections. In this work, it was revealed that most XDR isolates tended to develop stronger biofilms compared to MDR isolates, and it is suggested a direct relationship between XDR and biofilm formation capacity. Another study indicated that, in the KPC-positive group, the irp2, mrkD, and fimH virulence genes had a higher frequency than in the KPC-negative group (38). Another work, reported that the most of the K. pneumoniae isolates harbored both fimH-1 and mrkD virulence genes (39). Another study indicated that the irp2 and fimH genes were more detected among K. pneumoniae isolated (40). The genes of entB and mrkD were the most prevailing virulence genes (41). Our findings are in agreement with these studies. Therefore, the presence of genes of entB, magA, Irp2, fimH, and mrkD which are found in our survey, illustrates the importance of evaluating these virulence factors. It should be noted that, the results of these studies were similar, and the differences in results could be due to differences in the study population.

The results of this study demonstrated the prevalence of infections caused by β-lactamase-producing K. pneumoniae, which are biofilm producers among COVID-19 Patients. In the current study, all isolates produced strong and moderate biofilm. The results indicated that strong and moderate biofilm formation isolates need to address for new category of antibiotics. The effective antimicrobial activity of tigecycline to the bacteria that produce these enzymes may be efficient in faster and better treating patients which are hospitalized. The monitoring and control of hospital-acquired infections should be considered, to reduce the spread of MDR/XDR bacteria. These include surveillance systems to notice changes in drug resistance profile and etiology, setting experimental treatment guidelines based on the profiles, and proper instruction of healthcare workers regarding sanitation. Future studies should include more complex microbial communities residing in the Hospitals. Also, to the field of study using new antibiotics should be addressed.

ESBL

extended-spectrum-β-lactamase

CRE

Carbapenem-Resistant Enterobacteriaceae

CRKP

carbapenemase-producing Klebsiella pneumonia

BAL

bronchoalveolar lavage

TSA

trypticase soy agar

KPC

Klebsiella pneumoniae Carbapenemase.

Ethics approval and consent to participate

The current study was performed by approval of the Ethics Committee of Qazvin Medical University with approval number IR.QUMS.REC.1400.166. In addition, the committee approved the utilization of human samples. We confirm that written informed consent to participate was obtained from all of the participants in our study. We acquired permissions and/or licenses to access the clinical patient data used in our research from Qazvin University of Medical Sciences. Hospitals provided the clinical samples. Also, it should be noted that biological samples are handled by the authors in the present study. The adopted methods for handling human samples were carried out in accordance with relevant guidelines and regulations provided in the Declaration of Helsinki. The research protocol was approved by the Research Ethics Committee at the Qazvin Medical University, Iran.

Consent for publication

Not applicable

Availability of data and materials

Data were collected in an electronic data capture system and are available on request to the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

Author contribution

This work was carried out in collaboration between all authors. Farhad Nikkhahi conceptualised and designed the research study. Sara Rahimi, Mehdi Bakht and Amir peymani performed the microbiological tests and molecular procedures and wrote the manuscript. Zahra Farshadzadeh and Mohadeseh khakpour contributed to the manuscript final version, and supervised the research process. Hasan Ehteram and Ashraf Bakhshi Contributed to sampling. All authors read and approved the final manuscript. current manuscript has been read and approved by all the authors, that the requirements for authorship have been met, and each author believes that the manuscript represents honest work.

Acknowledgement

We gratefully thank the Shahid Beheshti hospital Kashan, Iran. This work would not have been possible without their support of them

(UK). NIfHaCE. managing suspected or confirmed pneumonia in adults in the community. London2021. 78 p.
Li J, Wang J, Yang Y, Cai P, Cao J, Cai X, et al. Etiology and antimicrobial resistance of secondary bacterial infections in patients hospitalized with COVID-19 in Wuhan, China: a retrospective analysis. Antimicrob Resist Infect Control. 2020;9(1):1–7.
Cox MJ, Loman N, Bogaert D, O'Grady J. Co-infections: potentially lethal and unexplored in COVID-19. The Lancet Microbe. 2020;1(1):e11.
Siu LK, Yeh K-M, Lin J-C, Fung C-P, Chang F-Y. Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis. 2012;12(11):881–7.
Galehdar M, Ghane M, Babaeekhou L. Co-occurrence of Carbapenemase-encoding Genes Among Klebsiella pneumoniae Clinical Isolates: Positive Relationship of bla NDM and bla SIM with Imipenem Resistance.Jundishapur Journal of Microbiology. 2021;14(2).
Ni W, Li G, Zhao J, Cui J, Wang R, Gao Z, et al. Use of Monte Carlo simulation to evaluate the efficacy of tigecycline and minocycline for the treatment of pneumonia due to carbapenemase-producing Klebsiella pneumoniae. Infect Dis. 2018;50(7):507–13.
O'Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annual Reviews in Microbiology. 2000;54(1):49–79.
Wang Z, Cai R, Wang G, Guo Z, Liu X, Guan Y, et al. Combination therapy of phage vB_KpnM_P-KP2 and gentamicin combats acute pneumonia caused by K47 serotype Klebsiella pneumoniae. Front Microbiol. 2021;12:926.
Clinical, Institute LS. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute Wayne, PA; 2017.
EUCAST T. European Committee on Antimicrobial Susceptibility Testing., Breakpoint tables for interpretation of MICs and zone diameters.European Society of Clinical Microbiology and Infectious Diseases Basel&#8230
Magiorakos A-P, Srinivasan A, Carey RB, Carmeli Y, Falagas M, Giske C, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81.
Mohd Khari FI, Karunakaran R, Rosli R, Tee Tay S. Genotypic and phenotypic detection of AmpC β-lactamases in Enterobacter spp. isolated from a teaching hospital in Malaysia. PLoS ONE. 2016;11(3):e0150643.
Stürenburg E, Lang M, Horstkotte MA, Laufs R, Mack D. Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum β-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria. J Antimicrob Chemother. 2004;54(5):870–5.
Edelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and molecular epidemiology of CTX-M extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob Agents Chemother. 2003;47(12):3724–32.
Rebelo AR, Bortolaia V, Kjeldgaard JS, Pedersen SK, Leekitcharoenphon P, Hansen IM, et al. Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes. Eurosurveillance. 2018;23(6):17–00672.
Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005;43(8):4178–82.
Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Tournebize R, Diancourt L, et al. Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS ONE. 2009;4(3):e4982.
Rahimi S, Farshadzadeh Z, Taheri B, Mohammadi M, Haghighi M-A, Bahador A. The relationship between antibiotic resistance phenotypes and biofilm formation capacity in clinical isolates of Acinetobacter baumannii. Jundishapur J Microbiol. 2018;11(8):1–7.
Marques C, Menezes J, Belas A, Aboim C, Cavaco-Silva P, Trigueiro G, et al. Klebsiella pneumoniae causing urinary tract infections in companion animals and humans: population structure, antimicrobial resistance and virulence genes. J Antimicrob Chemother. 2019;74(3):594–602.
Devanga Ragupathi NK, Muthuirulandi Sethuvel DP, Triplicane Dwarakanathan H, Murugan D, Umashankar Y, Monk PN et al. The influence of biofilms on carbapenem susceptibility and patient outcome in device associated K. pneumoniae infections: insights into phenotype vs genome-wide analysis and correlation.Frontiers in microbiology. 2020:3220.
Sader HS, Castanheira M, Flamm RK, Mendes RE, Farrell DJ, Jones RN. Tigecycline activity tested against carbapenem-resistant Enterobacteriaceae from 18 European nations: results from the SENTRY surveillance program (2010–2013). Diagn Microbiol Infect Dis. 2015;83(2):183–6.
Mostafavi SN, Rostami S, Nokhodian Z, Ataei B, Cheraghi A, Ataabadi P, et al. Antibacterial resistance patterns of Acinetobacter baumannii complex: The results of Isfahan Antimicrobial Resistance Surveillance-1 Program. Asian Pac J Trop Med. 2021;14(7):316.
Xie J, Wang T, Sun J, Chen S, Cai J, Zhang W, et al. Optimal tigecycline dosage regimen is urgently needed: results from a pharmacokinetic/pharmacodynamic analysis of tigecycline by Monte Carlo simulation. Int J Infect Dis. 2014;18:62–7.
Lee C-R, Lee JH, Park KS, Kim YB, Jeong BC, Lee SH. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods. Front Microbiol. 2016;7:895.
Ssekatawa K, Byarugaba DK, Wampande E, Ejobi F. A systematic review: the current status of carbapenem resistance in East Africa. BMC Res Notes. 2018;11(1):1–9.
Gheitani L, Fazeli H. Prevalence of bla VIM, bla IMP, and bla KPC genes among carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from Kurdistan and Isfahan hospitals, Iran. Res Mol Med. 2018;6(2):12–20.
Maleki N, Tahanasab Z, Mobasherizadeh S, Rezaei A, Faghri J. Prevalence of CTX-M and TEM β-lactamases in Klebsiella pneumoniae isolates from patients with urinary tract infection, Al-Zahra hospital, Isfahan, Iran. Advanced biomedical research. 2018;7.
Saeidi S, Alavi-Naini R, Shayan S. Antimicrobial susceptibility and distribution of tem and ctx-m genes among esbl-producing Klebsiella pneumoniae and Pseudomonas aeruginosa causing urinary tract infections. Zahedan J Res Med Sci. 2014;16(4):1–5.
Kiratisin P, Apisarnthanarak A, Laesripa C, Saifon P. Molecular characterization and epidemiology of extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates causing health care-associated infection in Thailand, where the CTX-M family is endemic. Antimicrob Agents Chemother. 2008;52(8):2818–24.
Rimal U, Thapa S, Maharjan R. Prevalence of Extended spectrum beta-lactamase producing Escherichia coli and Klebsiella species from urinary specimens of children attending Friendship International Children’s Hospital. Nepal J Biotechnol. 2017;5(1):32–8.
Dehshiri M, Khoramrooz SS, Zoladl M, Khosravani SA, Parhizgari N, Motazedian MH, et al. The frequency of Klebsiella pneumonia encoding genes for CTX-M, TEM-1 and SHV-1 extended-spectrum beta lactamases enzymes isolated from urinary tract infection. Ann Clin Microbiol Antimicrob. 2018;17(1):1–7.
Eftekhar F, Golalipoor MRM, Mansour N. Detection of Extended Spectrum Β -Lactamases in Urinary Iso- lates of Klebsiella pneumoniae in Relation to Bla SHV, Bla TEM and Bla CTX-M Gene Carriage Iranian J Publ Health,. 2012;41(3):127–32.
Coque T, Baquero F, Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Eurosurveillance. 2008;13(47):19044.
Pishtiwan AH, Khadija KM. Prevalence of blaTEM, blaSHV, and blaCTX-M genes among ESBL-producing Klebsiella pneumoniae and Escherichia coli isolated from thalassemia patients in Erbil,Iraq. Mediterranean journal of hematology and infectious diseases. 2019;11(1).
Ni W, Wei C, Zhou C, Zhao J, Liang B, Cui J, et al. Tigecycline-amikacin combination effectively suppresses the selection of resistance in clinical isolates of KPC-producing Klebsiella pneumoniae. Front Microbiol. 2016;7:1304.
DiPersio JR, Dowzicky MJ. Regional variations in multidrug resistance among Enterobacteriaceae in the USA and comparative activity of tigecycline, a new glycylcycline antimicrobial. Int J Antimicrob Agents. 2007;29(5):518–27.
Fan B, Wang C, Song X, Ding X, Wu L, Wu H, et al. Bacillus velezensis FZB42 in 2018: the gram-positive model strain for plant growth promotion and biocontrol. Front Microbiol. 2018;9:2491.
KUŞ H, ARSLAN U, DAĞI HT. Hastane Enfeksiyonu Etkeni Klebsiella pneumoniae İzolatlarında Çeşitli Virülans Faktörlerinin Araştırılması. Mikrobiyol Bul. 2017;51(4):329–39.
Ferreira RL, da Silva B, Rezende GS, Nakamura-Silva R, Pitondo-Silva A, Campanini EB, et al. High prevalence of multidrug-resistant Klebsiella pneumoniae harboring several virulence and β-lactamase encoding genes in a Brazilian intensive care unit. Front Microbiol. 2019;9:3198.
Hormozi B, Rashki A, Alipoor M, Najimi M. Frequency of Pathogenic Genes fimH، irp2، rmpA، allS and wcaG of Klebsiella pneumoniae Isolates by Multiplex-PCR Method. Iran J Med Microbiol. 2018;11(6):178–83.
Albasha AM, Abd-Alhalim S, Alshaib EF, Al-Hassan L, Altayb HN. Detection of several carbapenems resistant and virulence genes in classical and hyper-virulent strains of Klebsiella pneumoniae isolated from hospitalized neonates and adults in Khartoum. BMC Res Notes. 2020;13(1):1–7.
Gupta G, Tak V, Mathur P. Detection of AmpC β lactamases in gram-negative bacteria. J Lab physicians. 2014;6(01):001–6.
Lee Y-J, Huang C-H, Ilsan NA, Lee I-H, Huang T-W. Molecular epidemiology and characterization of carbapenem-resistant klebsiella pneumoniae isolated from urine at a teaching hospital in Taiwan. Microorganisms. 2021;9(2):271.

No competing interests reported.

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Assessment of the activity of tigecycline and colistin on biofilm producer Klebsiella pneumoniae isolated from COVID-19 patients, Iran

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Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

Bacterial strains

Antibiotic Susceptibility Testing

Phenotypic Screening of ESBL, AmpC betalactamase, and Carbapenemase Producer-K. pneumonia

Detection Of Esbl, Ampc, And Carbapenemase-related Genes

Multilocus Sequence Typing (Mlst)

Biofilm Formation Assays

Detection Of Virulence Genes

Statistical Analysis

Results

Antimicrobial Susceptibility

Genotypic Assays For Esbl, Ampc, And Carbapenemase

Molecular Typing

Assessment Of Biofilm Formation Capacity

Assessment Of Virulence Factors

The Correlation Between Biofilm Formation And Antibiotic Resistance Phenotypes

Association between the presence of virulence gene and biofilm formation

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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