First Detection of Klebsiella pneumoniae Isolate Co-Harboring Fosfomycin Resistance Gene fosA3 and bla ctx-m Among Gram Negative Urine Isolates in a Turkish Hospital

Abstract

The most common gram-negative pathogens in urinary tract infections are Escherichia coli and Klebsiella pneumoniae. Therapy that is often empirical relies on local antibiotic resistance data, hence monitorization of antimicrobial resistance periodically in each hospital is a requirement. In this study, antibiotic susceptibility profiles of consecutive urinary isolates (E. coli [n = 235] and K. pneumoniae [n = 56]) of adult patients collected between February 2018 and February 2019 from inpatients in Hacettepe University Hospital were assessed. Isolates resistant to fosfomycin (minimum inhibitory concentration >32 mg/L) were further investigated for the presence of fosA, fosA3, and fosC2. Fosfomycin susceptibility was determined by agar dilution method. Broth microdilution method was performed for amikacin, gentamicin, ceftazidime, ceftriaxone, meropenem, ciprofloxacin, tigecycline, trimethoprim/sulfamethoxazole (TMP/SMX), colistin, and piperacillin/tazobactam (PIP/TAZ). PCR method was employed to investigate fosA, fosA3, fosC2, and bla_CTX-M. Existence of fosA3 gene was confirmed by sequencing. Resistance rates to amikacin, gentamicin, ceftazidime, ceftriaxone, meropenem, ciprofloxacin, tigecycline, TMP/SMX, colistin, and PIP/TAZ were 2.7%, 18.5%, 25.4%, 33.0%, 3.4%, 45.4%, 2.4%, 43.6%, 6.2%, and 23.7%, respectively. Sixteen isolates (5.5%) were resistant to fosfomycin. Resistance was most frequently observed in K. pneumoniae (n = 9). fosA3 gene was detected in one fosfomycin-resistant K. pneumoniae isolate. This isolate also carried bla_CTX-M. fosC2 and fosA genes could not be detected in any of the isolates. In this study, we report for the first time the existence of fosA3 in Turkey and its association with the bla_CTX-M gene. As a result of increasing bla_CTX-M producing Enterobacterales isolates globally, increase in fosfomycin resistance may be expected in near future.

Introduction

Urinary tract infections (UTIs) are the most common infectious diseases in humans and represent a significant economic burden and a major public health problem.¹ UTI ranks fourth among hospital-acquired infections with a prevalence of 12.9% and gram-negative bacteria are the most common pathogens in hospital and community-acquired UTIs.²

The most common pathogen in UTIs is Escherichia coli followed by other species of Enterobacterales.³

Fosfomycin is a broad spectrum bactericidal antibiotic that was discovered in 1969 and with the development of antibiotic resistance in many gram-negative bacteria, especially carbapenemase producing Enterobacterales, it has gained importance as an alternative drug in uncomplicated UTIs.³ Fosfomycin exhibits good efficacy for the treatment of uncomplicated UTIs and it is approved as an oral regimen in many parts of the world.⁴

Resistance to fosfomycin can be due to multiple mechanisms; target site modification (MurA), modifying enzymes (FosA, FosB, and FosC) or decreased permeability (GlpT or UhpT).⁵ Although the geographic distribution of resistance factors is diverse, the most common resistance mechanism is plasmid-mediated Fos enzymes, particularly fosA3.^6,7

The aim of this study was to evaluate and compare the antimicrobial susceptibility profiles of fosfomycin and 10 other antibiotics against E. coli and Klebsiella pneumoniae from urine samples and identify the most common resistance mechanisms of fosfomycin.

Materials and Methods

Bacterial isolates

A total of 291 E. coli and K. pneumoniae isolates were obtained from urinary samples of adult inpatients. All isolates were recovered consecutively between February 2018 and February 2019 in Hacettepe University Hospitals Bacteriology Laboratory. Only one isolate from each patient was included in the study. Isolates were stored at −20°C in 10% glycerol broth until further testing. Microorganisms were identified using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker, Germany). We have received the approval by the hospital ethics committee for the use of samples for human patients. (Hacettepe University Non-Interventional Clinical Researches Ethics Board No. GO18/260).

Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC)s of fosfomycin were detected by agar dilution using cationic Mueller–Hinton agar (Oxoid; Thermo Scientific, UK) supplemented with 25 mg/L of glucose-6-phosphate (Sigma-Aldrich, Missouri).⁸ Antibiotic concentrations in agar plates ranged from 0.25 to 256 mg/L. Bacterial suspensions were prepared to achieve a final inoculum of 1 × 10⁴ CFU per spot using a multipoint inoculator (Abcam, Cambridge). The plates were incubated for 18 hours in ambient air at 35°C.

For microbroth dilution tests, amikacin, gentamicin, ceftazidime, ceftriaxone, meropenem, ciprofloxacin, tigecycline, trimethoprim/sulfomethoxazole (TMP/SMX), colistin, and piperacillin/tazobactam (PIP/TAZ) were supplied in powder forms (Sigma-Aldrich). All results were interpreted according to the EUCAST (2020; v 10.0) guidelines. Reference strains of E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and also mcr-1 positive E. coli NCTC 13846 were included as quality controls.

Molecular detection of bla_CTX-M and fosfomycinases

Each fosfomycin-resistant isolate was scanned for plasmid-encoded fosfomycin resistance genes (fosA, fosA3, and fosC2) by PCR. fosA and fosA3-carrying isolates were further screened for bla_CTX-M. Primers for these resistance genes are listed in Table 1.^9–11 The isolate that was positive for fosA3 and bla_CTX-M by PCR was confirmed by ABI 3730XL sequencer (Applied Biosystems, Foster City, CA) and compared with the GenBank DNA sequence database by using the genomic BLASTN program (available at www.ncbi.nlm.nih.gov/blast).

Table 1.

Primers Used for PCR Amplification of Fosfomycin Resistance and bla_CTX-M Genes

Gene names	Sequence	Amplicon size (bp)
fosA	F-5′-ATC TGT GGG TCT GCC TGT CGT-3′ R-5′-ATG CCC GCA TAG GGC TTCT-3′	271
fosA3	F-5′-GGC ATT TTA TCA GCA GT-3′ R-5′-AGA CCA TCC CCT TGT AG-3′	350
fosC2	F-5′-CGA GCC AAG ATT ACT GT-3′ R-5′-AAC GAT TCC AAA CGA CT-3′	196
bla _CTX-M	F-5′-TCT TCC AGA ATA AGG AAT CCC-3′ R-5′- CCG TTT CCG CTA TTA CAA AC-3′	909

Results

Bacterial isolates

Demographic characteristics of the patients and sample types according to pathogens are listed in Table 2.

Table 2.

Demographic Characteristics of the Patients and Sample Types According to Pathogens

	Escherichia coli (n = 235)		Klebsiella pneumoniae (n = 56)
	n	%	n	%
Gender
Female	186	79.1	38	67.9
Male	49	20.9	18	32.1
Clinical sample
Mid-stream urine	220	93.6	49	87.5
Nephrostomy	12	5.1	5	8.9
Catheter	3	1.3	2	3.6

Antimicrobial susceptibility testing

Susceptibility rates for fosfomycin and the comparator antibiotics are shown in Table 3. The highest resistance rate among E. coli isolates (n = 235) was detected against TMP/SMX (43.8%) and ciprofloxacin (43%). Overall, 16 (5.5%) isolates were resistant to fosfomycin. Resistance to fosfomycin was more frequent in K. pneumoniae (16.1%) than E. coli (3%). In K. pneumoniae, ciprofloxacin and PIP/TAZ were the agents with the highest resistance rates.

Table 3.

Susceptibility Rates of Fosfomycin and the Comparator Antibiotics

Antibiotic	Escherichia coli (n = 235)				Klebsiella pneumoniae (n = 56)
Antibiotic	S (%)	I (%)	R (%)	MIC range (mg/L)	S (%)	I (%)	R (%)	MIC range (mg/L)
FOF	228 (97.0)	—^a	7 (3.0)	0.25–>256	47 (83.9)	—^a	9 (16.1)	2–>256
AMK	227 (96.6)	6 (2.5)	2 (0.9)	<0.125–64	50 (89.3)	0	6 (10.7)	0.125–>256
GEN	194 (82.5)	2 (0.9)	39 (16.6)	<0.125–>256	41 (73.2)	0	15 (26.8)	<0.125–>256
CAZ	169 (72.0)	12 (5.0)	54 (23.0)	<0.125–>256	34 (60.7)	2 (3.6)	20 (35.7)	<0.125–>256
CRO	158 (67.2)	5 (2.1)	72 (30.7)	<0.125–>256	29 (51.8)	3 (5.4)	24 (42.8)	<0.125–>256
MEM	231 (98.3)	1 (0.4)	3 (1.3)	<0.125–>256	48 (85.7)	1 (1.8)	7 (12.5)	<0.125–>64
CIP	120 (51.0)	14 (6.0)	101 (43.0)	<0.015–128	23 (41.1)	2 (3.6)	31 (55.3)	<0,015–>256
TGC	229 (97.4)	2 (0.9)	4 (1.7)	<0.015–>32	50 (89.2)	3 (5.3)	3 (5.3)	<0.015–8
TMP/SMX	132 (56.2)	0	103 (43.8)	<0.015–>32	31 (55.4)	1 (1.8)	24 (42.8)	<0.015–>32
COL	223 (94.9)	—^a	12 (5.1)	<0.015–>32	50 (89.3)	—^a	6 (10.7)	<0.015–32
TZP	189 (80.4)	8 (3.4)	38 (16.2)	<0.125–>256	23 (41.1)	2 (3.6)	31 (55.3)	<0.125–>256

No intermediate value.

AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; COL, colistin; CRO, ceftriaxone; FOF, fosfomycin; GEN, gentamicin; I, Intermediate; MEM, meropenem; R, resistance; S, susceptible; TGC, tigecycline; TMP/SMX, trimethoprim/sulfamethoxazole; TZP, piperacillin/tazobactam.

PCR and sequencing

Among 16 fosfomycin-resistant isolates, fosA3 gene was detected in one K. pneumoniae isolate. This isolate also carried bla_CTX-M. (GenBank accession number for bla_CTX-M was MW750217 and for fosA MW750218) fosC2 gene could not be detected in any of the isolates.

Discussion

Fosfomycin is a cell wall-active antimicrobial agent found to be effective against E. coli, Citrobacter spp, Enterobacter spp, Klebsiella spp, Serratia spp, and Enterococcus faecalis related UTIs.^12–14 Previous studies have shown that fosfomycin can be an option for empirical therapy in uncomplicated UTI outpatients infected with isolates resistant to TMP/SMX, fluoroquinolones and cephalosporins obtained as a routine microbiology laboratory test result with automated system.¹⁵ According to our study, fosfomycin showed higher in vitro activity compared to other antimicrobial agents in E. coli isolates.

Overall rates of fosfomycin resistance in E. coli has remained low worldwide, at 1.2–3.5%^15,16 and 7.2–28.6% in Klebsiella spp.^17,18 In Asian countries, fosfomycin resistance rates are relatively higher than other countries. In a study from China, the rate of fosfomycin resistance was 6.7% in E. coli isolates and the main resistance mechanism was the presence of the fosA3 gene.¹⁹ In a study from Taiwan, fosfomycin had excellent activity against extended-spectrum beta-lactamases (ESBL) producing E. coli isolates, with a 4.5% resistance rate.²⁰

In Europe, resistance to fosfomycin in UTI isolates remain lower than Asia. Studies from Germany and France show fosfomycin resistance rates ∼1.0% in urinary isolates of E. coli.^21,22 In a recent study from our hospital, the resistance rate of fosfomycin was 2.7% in urinary E. coli isolates (n = 299).²³

Several fosfomycin resistance mechanisms have been described in E. coli, including reduced permeability, modification of the murA gene target, and modification of fosfomycin.²⁴ In E. coli, the plasmid-mediated fosfomycin resistance gene fosA3, which encodes a glutathione S-transferase, was first identified in a fosfomycin-resistant E. coli strain in Japan.⁷ In some studies, especially from Asia, the high transferability of bla_CTX-M and fosA3 through plasmids with identical replicon types further indicates that the two genes may be simultaneously disseminated by plasmids.^9,11 Lee et al.²⁵ concluded that common feature that the fosA3 gene, connected to bla_CTX-M through insertion sequences, was located between two IS26 elements oriented in the opposite direction, composing an IS26-composite transposon.

Results of various studies show that the clonal spread of the CTX-M-15 are linked with fosfomycin resistance.^26,27 In a study conducted in Korea,²⁵ resistance to fosfomycin was 7.1% in E. coli and 4.8% in K. pneumoniae. Of the 21 isolates resistant to fosfomycin, 7 (E. coli [n = 5] and K. pneumoniae [n = 2]) isolates harbored fosA3 and all of them co-harbored ESBLs bla_CTX-M-1 type or bla_CTX-M-9 type. There are limited studies to elucidate fosfomycin resistance mechanisms in our country. To our knowledge, this is the first report that shows the coexistence of fosA3 and bla_CTX-M gene in Turkey. The significance of this finding is that there is a high risk for their dissemination and suggests a critical need for close monitoring of such isolates.

Oral antibiotic options for the treatment of UTI, especially the ones caused by MDR isolates are limited. TMP/SMX and ciprofloxacin were commonly used to treat UTIs, but in recent years resistance rates of E. coli isolates to these drugs have increased significantly. According to the reports in the past decade, 10–15% resistance rate for TMP/SMX was reported in Japan,²⁸ 30% in both China and South Korea.^29,30 In Europe and the Mediterranean region, the resistance rates of E. coli isolates to TMP/SMX varied but were usually >15%.^31,32 In various reports from different countries, resistance to ciprofloxacin was reported as 15.1–45.0% in E. coli and 18.7–51.2% in K. pneumoniae.^2,15,33

In several recent studies from Turkey, resistance to ciprofloxacin was 29.5–70% and to TMP/SMX was 20.0–62.0%.^32,34,35 In our study, resistance rates of E. coli to ciprofloxacin and TMP/SMX were 43% and 43.8%, respectively, and these rates were 55.3% and 42.8%, respectively, in K. pneumoniae.

In a recent review of the global epidemiology, PIP/TAZ is reported to retain good activity against E. coli isolates but exhibits diminished activity in isolates of K. pneumoniae, especially in isolates producing ESBLs. In our study, in K. pneumoniae overall resistance to PIP/TAZ is 55.3%. It is noteworthy that this resistance rate is somewhat higher than in most of the published surveillance studies that report values between 4.2% and 30.3%.^36,37 In a recent study, resistance of K. pneumoniae isolates to PIP/TAZ (≥20%) was shown to be stable over the years among UTI isolates.³⁸ In another study from Asian countries, among ESBL-positive UTI isolates of K. pneumoniae, susceptibility to PIP/TAZ was <54%.³⁹

Rates of resistance to colistin, tigecycline, and meropenem remain low. A global survey reported colistin susceptibility rates of 97.0–99.0% in K. pneumoniae and E. coli isolates, respectively, in North America, Europe, Latin America, and Asia pacific.⁴⁰ Meropenem resistance ranged between 0% and 16.0%.^25,41 Tigecycline is not usually recommended for the treatment of UTIs due to low peak serum concentrations, limited excretion into urine, and subsequent development of resistance.⁴² Against E. coli and K. pneumoniae, we demonstrated lower resistance rates to colistin (5.1%, 10.7%), tigecycline (1.7%, 5.3%), and meropenem (1.3%, 12.5%) than other comparative agents, respectively.

As a limitation of this study, we were unable to identify potential and novel fosfomycin resistance genes as the whole genome sequencing could not performed, although we have performed sequencing for fosA3.

Conclusion

Fosfomycin has a good in vitro activity against urine E. coli and K. pneumoniae isolates in our hospital. Fosfomycin resistance was higher in K. pneumoniae isolates than E. coli. This is the first report from Turkey showing the existence of fosA3 with the bla_CTX-M gene. It indicates that fosfomycin resistance mechanisms may be associated with other resistance genes and should be examined periodically in locations where fosfomycin is increasingly used for empiric treatment of UTIs.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by a grant from the Hacettepe University Scientific Research Projects Commission project number THD-2018-17031.

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First Detection of Klebsiella pneumoniae Isolate Co-Harboring Fosfomycin Resistance Gene fosA3 and bla ctx-m Among Gram Negative Urine Isolates in a Turkish Hospital

Abstract

Introduction

Materials and Methods

Bacterial isolates

Antimicrobial susceptibility testing

Molecular detection of blaCTX-M and fosfomycinases

Results

Bacterial isolates

Antimicrobial susceptibility testing

PCR and sequencing

Discussion

Conclusion

Footnotes

Disclosure Statement

Funding Information

References

Molecular detection of bla_CTX-M and fosfomycinases