In the present study a total of 200 Klebsiella pneumoniae isolates were collected from patients with urinary tract infections (UTIs) in Tehran, Iran. Antibiotic resistance was determined by disk diffusion and broth dilution methods. Detection of extended-spectrum β-lactamases (ESBLs) and AmpCs was performed using phenotypic tests. Polymerase chain reaction (PCR) was applied to detect the ESBL, AmpC, and integron genes. Analysis of AmpC and cassette arrays of integron genes was performed using DNA sequencing. Plasmids were analyzed by PCR-based replicon typing and conjugation. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were applied to explore the genomic relatedness among the isolates. The highest levels of resistance were observed against ampicillin (100%), followed by piperacillin (57.5%), ceftazidime (46%), trimethoprim/sulfamethoxazole (44%), ciprofloxacin (32.5%), and imipenem (19%). Approximately, 66.5% of isolates harbored at least one of the beta-lactamase genes (blaTEM, blaSHV, blaCTX-M, and blaOXA-1). In addition, 22.5% of isolates carried at least one of the AmpC genes including blaDHA and blaCIT. Integron class I was the most prevalent integron among resistant isolates. According to the results of replicon typing, IncFII, IncL/M, and IncA/C were the most frequent replicons, respectively. All selected isolates were able to transfer blaCTX-M, also two isolates transferred the blaDHA-1 gene to Escherichia coli K12 through conjugation. Finally, 21 isolates were categorized into 4 pulsotypes and 11 unique clusters in PFGE. MLST identified ST147 and ST11 sequence types but ST147 was the most prevalent in the current study.
Ding Y, Wang H, Pu S, Huang S, Niu S. Resistance trends of Klebsiella pneumoniae causing urinary tract infections in Chongqing, 2011–2019. Infect Drug Resist 2021; 14: 475–481.
Shivaee A, Meskini M, Shahbazi S, Zargar M. Prevalence of flmA, flmH, mrkA, ecpA, and mrkD virulence genes affecting biofilm formation in clinical isolates of K. pneumonia. KAUMS J (FEYZ) 2019; 23: 168–176.
Xiong Y, Zhang C, Gao W, Ma Y, Zhang Q, Han Y, et al. Genetic diversity and co-prevalence of ESBLs and PMQR genes among plasmid-mediated AmpC β-lactamase-producing Klebsiella pneumoniae isolates causing urinary tract infection. J Antibioti (Tokyo) 2021; 74: 397–406.
Livermore DM. β-Lactamases-the threat renews. Curr Protein Pept Sci 2009; 10: 397–400.
Shahbazi S, Karam MRA, Habibi M, Talebi A, Bouzari S. Distribution of extended-spectrum β-lactam, quinolone and carbapenem resistance genes, and genetic diversity among uropathogenic Escherichia coli isolates in Tehran, Iran. J Glob Antimicrob Resist 2018; 14: 118–125.
Shivaee A, Shahbazi S, Soltani A, Ahadi E. Evaluation of the prevalence of broad-spectrum beta-lactamases (ESBLs) and carbapenemase genes in Klebsiella pneumoniae strains isolated from burn wounds in patients referred to Shahid Motahari Hospital in Tehran. Med Sci J Islam Azad Univ 2019; 29: 232–239.
Ribeiro T, Novais Â, Rodrigues C, Nascimento R, Freitas F, Machado E, et al. Dynamics of clonal and plasmid backgrounds of Enterobacteriaceae producing acquired AmpC in Portuguese clinical settings over time. Int J Antimicrob Agents 2019; 53: 650–656.
Maeyama Y, Taniguchi Y, Hayashi W, Ohsaki Y, Osaka S, Koide S, et al. Prevalence of ESBL/AmpC genes and specific clones among the third-generation cephalosporin-resistant Enterobacteriaceae from canine and feline clinical specimens in Japan. Vet Microbiol 2018; 216: 183–189.
Dziri O, Dziri R, Maraoub A, Chouchani C. First report of SHV-148-Type ESBL and CMY-42-type AmpC β-lactamase in Klebsiella pneumoniae clinical isolates in Tunisia. Microb Drug Resist 2018; 24: 1483–1488.
Rizi KS, Mosavat A, Youssefi M, Jamehdar SA, Ghazvini K, Safdari H, et al. High prevalence of blaCMY AmpC beta-lactamase in ESBL co-producing Escherichia coli and Klebsiella spp. clinical isolates in the northeast of Iran. J Glob Antimicrob Resist 2020; 22: 477–482.
Hu F, Wu W, Ye X, Xu X, Zhu D. The molecular characteristics of cefepime-susceptible Escherichia coli and Klebsiella spp. isolates with a positive β-lactamase screening test result but negative confirmation. Eur J Clin Microbiol Infect Dis 2010; 29: 1297–1299.
Pfeifer Y, Cullik A, Witte W. Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int J Med Microbiol 2010; 300: 371–379.
Meini S, Tascini C, Cei M, Sozio E, Rossolini GM. AmpC β-lactamase-producing Enterobacterales: what a clinician should know. Infection 2019; 47: 363–375.
Rodríguez-Martínez JM, Machuca J, Cano ME, Calvo J, Martinez-Martinez L, Pascual A. Plasmid-mediated quinolone resistance: two decades on. Drug Resist Updat 2016; 29: 13–29.
Shivaee A, Mirshekar M. Association between ESBLs genes and quinolone resistance in uropathogenic Escherichia coli isolated from patients with urinary tract infection. Infect Epidemiol Microbiol 2019; 5: 15–23.
Denisuik AJ, Lagacé-Wiens PR, Pitout JD, Mulvey MR, Simner PJ, Tailor F, et al. Molecular epidemiology of extended-spectrum β-lactamase-, AmpC β-lactamase-and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolated from Canadian hospitals over a 5 year period: canward 2007–11. J Antimicrob Chemother 2013; 68(Suppl 1): i57–i65.
Wayne P. Performance standards for antimicrobial susceptibility testing: twenty fifth international supplement M100-S25. Clinical and Laboratory Standards Institute; 2015.
Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40: 2153–2162.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–228.
Nesporova K, Valcek A, Papagiannitsis C, Kutilova I, Jamborova I, Davidova-Gerzova L, et al. Multi-drug resistant plasmids with ESBL/AmpC and mcr-5.1 in Paraguayan poultry farms: the linkage of antibiotic resistance and hatcheries. Microorganisms 2021; 9: 866.
Zhan L, Wang S, Guo Y, Jin Y, Duan J, Hao Z, et al. Outbreak by hypermucoviscous Klebsiella pneumoniae ST11 isolates with carbapenem resistance in a tertiary hospital in China. Front Cel Infect Microbiol 2017; 7: 182.
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233–2239.
Lim T-P, Cai Y, Hong Y, Chan ECY, Suranthran S, Teo JQ-M, et al. In vitro pharmacodynamics of various antibiotics in combination against extensively drug-resistant Klebsiella pneumoniae. Antimicrob Agents Chemother 2015; 59: 2515–2524.
Shivaee A, Mohammadzadeh R, Shahbazi S, Pardakhtchi E, Ohadi E, Kalani BS. Time-variable expression levels of mazF, atlE, sdrH, and bap genes during biofilm formation in Staphylococcus epidermidis. Acta Microbiol Immunol Hung 2019; 66: 499–508.
El Bouamri M, Arsalane L, El Kamouni Y, Zouhair S. Antimicrobial susceptibility of urinary Klebsiella pneumoniae and the emergence of carbapenem-resistant strains: a retrospective study from a university hospital in Morocco, North Africa. Afr J Urol 2015; 21: 36–40.
Goodarzi NN, Fereshteh S, Sabzi S, Shahbazi S, Badmasti F. Construction of a chimeric FliC including epitopes of OmpA and OmpK36 as a multi-epitope vaccine against Klebsiella pneumonia. Health Biothechnology and Biopharma 2021; 5: 44–60.
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. 28.
Beyene D, Bitew A, Fantew S, Mihret A, Evans M. Multidrug-resistant profile and prevalence of extended spectrum β-lactamase and carbapenemase production in fermentative Gram-negative bacilli recovered from patients and specimens referred to National Reference Laboratory, Addis Ababa, Ethiopia. PloS one 2019; 14, e0222911.
Dahle KW, Korgenski EK, Hersh AL, Srivastava R, Gesteland PH. Clinical value of an ambulatory-based antibiogram for uropathogens in children. J Pediatr Infect Dis Soc 2012; 1: 333–336.
Ghasemi Y, Archin T, Kargar M, Mohkam M. A simple multiplex PCR for assessing prevalence of extended-spectrum β-lactamases producing Klebsiella pneumoniae in Intensive Care Units of a referral hospital in Shiraz, Iran. Asian Pac J Trop Med 2013; 6: 703–708.
Derakhshan S, Najar Peerayeh S, Bakhshi B. Association between presence of virulence genes and antibiotic resistance in clinical Klebsiella pneumoniae isolates. Lab Med 2016; 47: 306–311.
Ranjbar R, Memariani H, Sorouri R, Memariani M. Distribution of virulence genes and genotyping of CTX-M-15-producing Klebsiella pneumoniae isolated from patients with community-acquired urinary tract infection (CA-UTI). Microb Pathog 2016; 100: 244–249.
Japoni A, Gudarzi M, Farshad S, Basiri E, Ziyaeyan M, Alborzi A, et al. Assay for integrons and pattern of antibiotic resistance in clinical Escherichia coli strains by PCR-RFLP in Southern Iran. Jpn J Infect Dis 2008; 61: 85–88.
Renuart AJ, Goldfarb DM, Mokomane M, Tawanana EO, Narasimhamurthy M, Steenhoff AP, et al. Microbiology of urinary tract infections in Gaborone, Botswana. PLoS One 2013; 8, e57776.
Dotis J, Printza N, Marneri A, Gidaris D, Papachristou F. Urinary tract infections caused by extended-spectrum beta-lactamase-producing bacteria in children: a matched case-control study. Turk J Pediatr 2013; 55: 571–574.
Gürntke S, Kohler C, Steinmetz I, Pfeifer Y, Eller C, Gastmeier P, et al. Molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae from bloodstream infections and risk factors for mortality. J Infect Chemother 2014; 20: 817–819.
Feizabadi MM, Mahamadi-Yeganeh S, Mirsalehian A, Mirafshar S-M, Mahboobi M, Nili F, et al. Genetic characterization of ESBL producing strains of Klebsiella pneumoniae from Tehran hospitals. J Infect Dev Ctries 2010; 4: 609–615.
Lukac PJ, Bonomo RA, Logan LK. Extended-spectrum β-lactamase–producing Enterobacteriaceae in children: old foe, emerging threat. Clin Infect Dis 2015; 60: 1389–1397.
Tian G-B, Garcia J, Adams-Haduch JM, Evangelista JP, Destura RV, Wang H-N, et al. CTX-M as the predominant extended-spectrum β-lactamases among Enterobacteriaceae in Manila, Philippines. J Antimicrob Chemother 2010; 65: 584–586.
Grover S, Sharma M, Chattopadhya D, Kapoor H, Pasha S, Singh G. Phenotypic and genotypic detection of ESBL mediated cephalosporin resistance in Klebsiella pneumoniae: emergence of high resistance against cefepime, the fourth generation cephalosporin. J Infect 2006; 53: 279–288.
Lan NPH, Hien NH, Phuong TLT, Thanh DP, Thieu NTV, Ngoc DTT, et al. Phenotypic and genotypic characteristics of ESBL and AmpC producing organisms associated with bacteraemia in Ho Chi Minh City. Vietnam Antimicrob Resist Infect Control 2017; 6: 105.
Nakano R, Okamoto R, Nagano N, Inoue M. Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase bla CMY-4 promoted by insertion sequence ISEcp1. J Infect Chemother 2007; 13: 18–23.
Yamasaki K, Komatsu M, Abe N, Fukuda S, Miyamoto Y, Higuchi T, et al. Laboratory surveillance for prospective plasmid-mediated AmpC β-lactamases in the Kinki region of Japan. J Clin Microbiol 2010; 48: 3267–3273.
Mata C, Miró E, Alvarado A, Garcillán-Barcia MP, Toleman M, Walsh TR, et al. Plasmid typing and genetic context of AmpC β-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: findings from a Spanish hospital 1999–2007. J Antimicrob Chemother 2011; 67: 115–122.
Japoni-Nejad A, Ghaznavi-Rad E, van Belkum A. Characterization of plasmid-mediated AmpC and carbapenemases among Iranain nosocomial isolates of Klebsiella pneumoniae using phenotyping and genotyping methods. Osong Public Health Res Perspect 2014; 5: 333–338.
Ghanavati R, Darban-Sarokhalil D, Navab-Moghadam F, Kazemian H, Irajian G, Razavi S. First report of coexistence of AmpC beta-lactamase genes in Klebsiella pneumoniae strains isolated from burn patients. Acta Microbiol Immunol Hung 2017; 64: 455–462.
Heinz E, Ejaz H, Scott JB, Wang N, Gujaran S, Pickard D, et al. Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1. Sci Rep 2019; 9: 2392.
Alonso N, Miró E, Pascual V, Rivera A, Simó M, Garcia MC, et al. Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates. Int J Antimicrob Agents 2016; 47: 62–68.
Caliskan E, Say CU, Dulger G, Kilincel O, Ankarali H, Sahin I. Investigation of plasmid mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae isolates by phenotypic and genotypic. J Pak Med Assoc 2019; 69: 834–839.
Collis CM, Kim MJ, Stokes H, Hall RM. Integron‐encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59‐be site. Mol Microbiol 2002; 46: 1415–1427.
Xu X, Li X, Luo M, Liu P, Su K, Qing Y, et al. Molecular characterisations of integrons in clinical isolates of Klebsiella pneumoniae in a Chinese tertiary hospital. Microb Pathog 2017; 104: 164–170.
Canal N, Meneghetti KL, Almeida CPd, Bastos MdR, Otton LM, Corção G. Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water. Braz J Microbiol 2016; 47: 337–344.
Mobarak-Qamsari M, Ashayeri-Panah M, Eftekhar F, Feizabadi MM. Integron mediated multidrug resistance in extended spectrum beta-lactamase producing clinical isolates of Klebsiella pneumoniae. Braz J Microbiol 2013; 44: 849–854.
Firoozeh F, Mahluji Z, Khorshidi A, Zibaei M. Molecular characterization of class 1, 2 and 3 integrons in clinical multi-drug resistant Klebsiella pneumoniae isolates. Antimicrob Resist Infect Control 2019; 8: 59.
Cao X, Xu X, Zhang Z, Shen H, Chen J, Zhang K. Molecular characterization of clinical multidrug-resistant Klebsiella pneumoniae isolates. Ann Clin Microbiol Antimicrob 2014; 13: 16.
Li B, Hu Y, Wang Q, Yi Y, Woo PC, Jing H, et al. Structural diversity of class 1 integrons and their associated gene cassettes in Klebsiella pneumoniae isolates from a hospital in China. PloS one 2013; 8, e75805.
Adams-Sapper S, Sergeevna-Selezneva J, Tartof S, Raphael E, Diep BA, Perdreau-Remington F, et al. Globally dispersed mobile drug-resistance genes in Gram-negative bacterial isolates from patients with bloodstream infections in a US urban general hospital. J Med Microbiol 2012; 61: 968–974.
Aghamohammad S, Badmasti F, Solgi H, Aminzadeh Z, Khodabandelo Z, Shahcheraghi F. First report of extended-spectrum betalactamase-producing Klebsiella pneumoniae among fecal carriage in Iran: high diversity of clonal relatedness and virulence factor profiles. Microbl Drug Resist 2020; 26: 261–269.
Carattoli A. Plasmids in Gram negatives: molecular typing of resistance plasmids. Int J Med Microbiol 2011; 301: 654–658.
Ho P, Lo W, Yeung M, Li Z, Chan J, Chow K, et al. Dissemination of pHK01-like incompatibility group IncFII plasmids encoding CTX-M-14 in Escherichia coli from human and animal sources. Vet Microbiol 2012; 158: 172–179.
Habeeb MA, Haque A, Nematzadeh S, Iversen A, Giske CG. High prevalence of 16S rRNA methylase RmtB among CTX-M extended-spectrum β-lactamase-producing Klebsiella pneumoniae from Islamabad, Pakistan. Int J Antimicrob Agents 2013; 41: 524–526.
Naseer U, Sundsfjord A. The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb Drug Resist 2011; 17: 83–97.
Oteo J, Cuevas O, López-Rodríguez I, Banderas-Florido A, Vindel A, Pérez-Vázquez M, et al. Emergence of CTX-M-15-producing Klebsiella pneumoniae of multilocus sequence types 1, 11, 14, 17, 20, 35 and 36 as pathogens and colonizers in newborns and adults. J Antimicrob Chemother 2009; 64: 524–528.
Cullik A, Pfeifer Y, Prager R, Baum Hv, Witte W. A novel IS26 structure is surrounding blaCTX-M genes in different plasmids of German clinical isolates of Escherichia coli. J Med Microbiol 2010; 59: 580–587.
Carattoli A, Villa L, Poirel L, Bonnin RA, Nordmann P. Evolution of IncA/C blaCMY-2-carrying plasmids by acquisition of the blaNDM-1 carbapenemase gene. Antimicrob Agents Chemother 2012; 56: 783–786.
Brañas P, Villa J, Viedma E, Mingorance J, Orellana MA, Chaves F. Molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae in a hospital in Madrid: successful establishment of an OXA-48 ST11 clone. Int J Antimicrob Agents 2015; 46: 111–116.
De Laveleye M, Huang T-D, Bogaerts P, Berhin C, Bauraing C, Sacré P, et al. Increasing incidence of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Belgian hospitals. Eur J Clin Microbiol Infect Dis 2017; 36: 139–146.
Solgi H, Badmasti F, Giske CG, Aghamohammad S, Shahcheraghi F. Molecular epidemiology of NDM-1-and OXA-48-producing Klebsiella pneumoniae in an Iranian hospital: clonal dissemination of ST11 and ST893. J Antimicrob Chemother 2018; 73: 1517–1524.
Edelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and molecular epidemiology of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob Agents Chemother 2003; 47: 3724–3732.
Lim KT, Yasin R, Yeo CC, Puthucheary S, Thong KL. Characterization of multidrug resistant ESBL-producing Escherichia coli isolates from hospitals in Malaysia. J Biomed Biotechnol 2009; 2009, 165637.
Sugumar M, Kumar KM, Manoharan A, Anbarasu A, Ramaiah S. Detection of OXA-1 beta-lactamase gene of Klebsiella pneumoniae from blood stream infections (BSI) by conventional PCR and in-silico analysis to understand the mechanism of OXA mediated resistance. PLoS One 2014; 9, e91800.
Sun G, Yi M, Shao C, Ma J, Zhang Q, Shao S. Novel class 1 integrons in multi-drug resistant isolates from eastern China. Indian J Microbiol 2014; 54: 227–231.
Levesque C, Piche L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995; 39: 185–191.