Recently, emergence of carbapenem-resistance, in particular due to Klebsiella pneumoniae carbapenemase (KPC), was observed among K. pneumoniae causing urinary tract infections in Croatia. The aim of the study was to characterize, antimicrobial susceptibility, carbapenem resistance, virulence traits and plasmid types of the urinary KPC positive isolates of K. pneumoniae. The antimicrobial susceptibility to a wide range of antibiotics was determined by broth microdilution method. The transferability of meropenem resistance was determined by conjugation (broth mating method) employing Escherichia coli J63 strain resistant to sodium azide. Genes encoding broad and extended-spectrum β-lactamases, plasmid-mediated AmpC β-lactamases, group A and B carbapenemases, and carbapenem hydrolyzing oxacillinases (blaOXA-48like), respectively, were determined by Polymerase chain reaction (PCR). In total 30 KPC-positive K. pneumoniae urinary isolates collected from different regions of Croatia were analysed. The isolates were uniformly resistant to all tested antibiotics except for variable susceptibility to gentamicin, sulphamethoxazole/trimethoprim, and colistin, respectively. Four isolates were resistant to colistin with MICs values ranging from 4 to 16 mg/L. All tested isolates were susceptible to ceftazidime/avibactam. Sixteen isolates transferred meropenem resistance to E. coli recipient strain by conjugation. Other resistance markers were not co-transferred. PCR was positive for blaKPC and blaSHV genes in all isolates whereas 13 isolates tested positive also for blaTEM genes. PCR based replicon typing (PBRT) revealed the presence of FIIs in 13 and FIA plasmid in two strains. The study showed dissemination of KPC-producing K. pneumoniae in urinary isolates, posing a new epidemiological and treatment challenge. Sulphamethoxazole/trimethoprim, colistin, and ceftazidime/avibactam remain so far, as the therapeutic options.
Emergence of carbapenemase-producing Klebsiella pneumoniae isolates has become serious problem worldwide. K. pneumoniae produces several carbapenemases belonging to Ambler class A serin β-lactamases K. pneumoniae carbapenemase (KPC), class B metallo-β-lactamases (MBL) of the IMP, VIM or NDM family and OXA-48-like β-lactamases belonging to the class D (Nordmann et al. 2019; Bonomo et al. 2018). KPC-1/2 was initially found in a carbapenem-resistant strain in North Carolina in 1996. Later, it spread along the East Coast of USA and then it disseminated worldwide. In Europe, the particularly high rates of KPC carbapenemases were reported in Poland, Greece, Italy and Israel (Nordmann et al. 2019).
The first KPC-positive K. pneumoniae in Croatia was reported in 2011 (Bedenić et al. 2012). In a subsequent year, it was followed by a remarkable increase in the number of carbapenem-resistant isolates. This observation gave rise to a multicentre study on carbapenem-resistance in Enterobacteriaceae from Croatia, conducted in 2011–2012 which revealed the predominance of the VIM-1- metallo-β-lactamase and only sporadic occurrence of KPC-2 carbapenemase (Zujić Atalić et al. 2014). The first outbreak of infections in Croatian hospital due to KPC-producing K. pneumoniae was reported in 2015 (Bedenić et al. 2015). The multicentre study on KPC-producing K. pneumoniae was published in 2016 and demonstrated the rapid spread of KPC-2 in northwest geographic region, while southern, coastal regions of Croatia, were spared of KPC, until recently (Jelić et al. 2016). Despite several publications on KPC carbapenemases in Croatia, there are no reports on the characterization of KPC β-lactamases originated from urinary isolates. The aim of this study was the analysis of the molecular epidemiology and mechanisms of antibiotic resistance and virulence in emerging KPC-producing K. pneumoniae isolates associated with urinary tract infections originating from three different geographical regions in Croatia.
Material and methods
Isolates and patients
In total 30 K. pneumoniae isolates were collected from urine samples, originating form three centers in Croatia which participated in the study in the period from January 2014 to December 2019: University Hospital Centre Split (1,600-bed centre in southeast, coastal region, covering 0.5 million-population), General Hospital Dubrovnik (300-bed hospital in southern coastal region covering 100.000-population), and Andrija Štampar Teaching Public Health Institute covering outpatient population and long-term care facilities (LTCF) in Zagreb County (1 million-population). All patients had significant bacteriuria and pyuria (> 5 white blood cells per high power field or positive leukocyte esterase) with > 105 CFU/mL and K. pneumoniae in pure culture. The isolates were identified to the species level by Vitek 2 or MALDI-TOF, depending on the laboratory where they were isolated. The screening of KPC carbapenemase was done using immunochromatografic assay RESIST-4 O.K.N.V. (Coris Bioconcept, Belgium).
Laboratory analysis of the isolates
The antimicrobial susceptibility to amoxicillin alone and combined with clavulanate, piperacillin/tazobactam, cefazoline, expanded spectrum cephalosporins (ESC: ceftazidime, cefotaxime, ceftriaxone), cefepime, imipenem, meropenem, ertapenem, gentamicin, ciprofloxacin, cotrimoxazole, and colistin was determined by broth microdilution method according to CLSI guidelines except for colistin which was done according to EUCAS (European Committee for Antimicrobial Susceptibility Testing 2019); agar dilution was used to test for susceptibility to fosfomycin; and E-test (bioMerieux, France) was used to test susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam. According to susceptibility test results, the isolates were classified as multi-drug resistant (MDR) or extensively drug-resistant, as described previously by Magiorakos et al. (2012).
The double disk synergy test (DDST) (Jarlier et al 1988) and the CLSI combined disk test with addition of clavulanic acid were performed to detect ESBLs (CLSI. 2018). Plasmid-mediated AmpC β-lactamases were detected by combined disk test using cephalosporin disks with 3-aminophenylboronic acid (PBA) (Coudron et al. 2005), while combined disk tests with imipenem and meropenem alone or combined with PBA, 0.1 M EDTA or both to screen for KPC, MBLs, or simultaneous production of KPC and MBL, respectively (Pasteran et al. 2009; Kim et al 2007).
The transferability of meropenem and cefotaxime resistance was determined by conjugation (broth mating method) at 35 °C employing E. coli J65 recipient strain resistant to sodium-azide (Elwel and Falkow 1986).
Polymerase chain reaction targeting genes conferring resistance to β-lactams, including broad spectrum and extended-spectrum β-lactamases (blaSHV, blaTEM, blaCTX-M, |blaOXA-9|, |blaOXA-1| and blaPER-1) plasmid-mediated AmpC β-lactamases (pAmpC), class A (blaKPC, blaSME, blaIMI, blaNMC), class B carbapenemases (blaVIM, blaIMP and blaNDM), carbapenem hydrolyzing oxacillinases (blaOXA-48-like), and fluoroquinolone resistance genes (qnrA, qnrB, qnrS), was carried out using protocols and conditions as described previously (Nüesch-Inderbinen et al. 1996, Arlet et al. 1995, Woodford et al. 2004, Pagani et al. 2004, Perez-Perez and Hanson. 2002, Poirel et al. 2011. Robiscek et al. 2006, Woodford et al. 2006). CTX-M group β-lactamases was detected by multiplex Polymerase chain reaction (PCR) (Saladin et al. 2002). Genetic context of blaCTX-M genes was determined by PCR mapping with forward primer for ISEcpI and IS26 combined with primer MA-3 (universal reverse primer for blaCTX-M genes) (Cannateli et al. 2014). PCR was used to analyse to detect plasmid encoded colistin resistance genes mcr-1 and mcr-2 in one isolate with reduced susceptibility to colistin (Liu Liu et al. 2016; Huang et al. 2015). Six representative SHV and TEM amplicons were subjected to sequencing on both strands in Eurofin (Graz, Austria).
Plasmids were extracted from donor strains and their respective transconjugants with Qiagen mini kit (QIAGEN Hamburg, Germany) according to the manufacturer’s instructions. PCR-based replicon typing (PBRT) (Carattoli et al. 2005, 2015) was applied to determine the plasmid content of the tested strains. Positive control strains for PBRT were kindly provided by dr. A. Carattoli (Instituto Superiore di Sanità, Rome, Italy).
String test was attempted by stretching a mucoviscous string from the colony using a standard bacteriologic loop, as described previously (Yao et al. 2015). If a viscous string > 5 mm was formed, isolate was defined as hypermucoviscous.
The production of hemolysin was tested on human blood agar plate and was considered positive when bacteria were stabbed with a sterile straight wire into 5% human blood agar, and after 18–24 h of incubation at 37 ºC, a clearing zone was observe (Bevivino et al., 2002).
For serum sensitivity testing blood was obtained by venipuncture from three healthy volunteers and was allowed to clot at room temperature for 30 min and overnight at 4 °C. After centrifugation at 1,000g for 15 min at 4 °C, serum was removed and pooled. A portion of the pooled serum was decomplemented by heating at 56 °C for 30 min and used as test controls. Bacterial susceptibility to serum killing was measured by assessing regrowth after incubation in normal human serum according to Schiller and Hatch method (Schiller and Hatch, 1983).
Bacterial isolates and epidemiological data
In total 30 non-duplicate K. pneumoniae isolates producing KPC β-lactamase were analysed (20 from Split, five from Dubrovnik, and five from Zagreb). Majority of isolates were obtained from the middle-catch urine, while six samples were catheter-urines. Five out of 30 patients were outpatients, 5 resided in nursing homes, while the remaining 20 were hospitalized (7 in ICUs, 8 in Departments of Internal Medicine, 3 in surgical wards and 2 in the Department of Infectious Diseases). There were 17 male and 13 female patients. Between September 2018 and March 2019 there were in total 271 urinary isolates of K. pneumoniae in University Hospital Split and the rate of KPC positivity was 15.8% (43/271). Between September 2018 and March 2019 there were in total 3090 urinary isolates of K. pneumoniae in University Hospital Split and the rate of KPC positivity was 12,2% (377/3090). Public Health Institute in Dubrovnik reported 485 urinary tract K. pneumoniae isolates between January and December 2019. There were seven carbapenem resistant isolates (1.4%) and five (1.03%) identified as KPC positive. Andrija Štampar Public Health Institute in Zagreb had 8110 urinary tract K. pneumoniae isolates in the period from January 2014 to December 2019 with 252 (3,1%) carbapenem resistant and 7 (0.08%) confirmed as KPC producing organisms.
Laboratory analysis of the isolates
The isolates were uniformly resistant to amoxicillin alone and combined with clavulanic acid, piperacillin/tazobactam, cefazoline, cefuroxime, expanded spectrum cephalosporins, meropenem, ertapenem, ceftzolozane/tazobactam and ciprofloxacin. High resistance rates were observed for cefepime and imipenem—97% (29/30), as well as for gentamicin—87% (26/30) as shown in Table 1. Interestingly, the hospital isolates exhibited high level resistance to gentamicin (MIC > 128 mg/L) whereas three LTCF isolates showed susceptibility with MICs of 1 mg/L or less. Colistin and sulphamethoxazole/trimethoprim preserved good activity with 87% (26/30) and 93% (28/30) of susceptible isolates, respectively. Colistin resistance was identified in four isolates; three from Dubrovnik (3/5–60%) and one from Split (1/20–5%). Inhibitor-based test with meropenem and PBA, was positive in all isolates which was consistent with the production of KPC. MBLs were excluded based on the lack of synergy with EDTA. Combined disk test with clavulanic acid tested positive in all LTCF and one hospital isolate, with the augmentation of the inhibition zone around cephalosporin disks ranging from 5 to 10 mm, confirming the production of ESBL (Table 1). All isolates tested negative in combined disk test with cephalosporins and PBA excluding the production of plasmid-mediated AmpC β-lactamase.
Sixteen isolates transferred meropenem resistance to E. coli recipient strain by conjugation with the frequency ranging from 7.8 × 10–8 to 4.2 × 10–2. Resistance markers to gentamicin and ciprofloxacin were not co-transferred alongside with meropenem resistance. Cefotaxime resistance was not transferable.
Polymerase chain reaction was positive for blaKPC and blaSHV genes in all isolates whereas thirteen isolates tested positive also for blaTEM genes. Sequencing revealed blaKPC-2, blaSHV-1, blaSHV-11 (two isolates) and blaTEM-1 genes. Sequencing revealed blaSHV-1 genes in strains UR 14,867, UR 14,937, UR 22,199, and 2982/12. blaSHV-11 genes were detected in strains 22,668/2717 and 50,760. Whereas blaTEM-1 genes were identified in isolates: UR16948, UR18838, UR2428, UR2834, UR3147 and UR4281. Five out of six ESBL-positive isolates were found to harbour blaCTX-M-9 genes, whereas blaSHV-12 gene was found in one remaining ESBL-positive isolate (Table 1). Insertion sequences IS26 and ISEcpI were not found upstream of the blaCTX-M genes. Plasmid-mediated mcr genes were not found in colistin resistant isolate. Qnr genes encoding reduced susceptibility to fluoroquinolones were not found. Transconjugants were positive for blaKPC-2 genes as their respective donors.
Polymerase chain reaction-based replicon typing revealed the presence of FIIS in 13 and FIA plasmid in 2 isolates. The transconjugant strains obtained with meropenem as selective agents were also positive for FIIS plasmids.
String test was positive in 16 whereas hemolysin production was detected in 5 isolates. All isolates demonstrated resistance to serum bactericidal activity as shown in Table 2.
At the national level, the spread of carbapenemase-producing Enterobacteriaceae in Croatia, began in 2014 with VIM-1 metallo-β-lactamase and only sporadic occurrence of KPC-2 carbapenemase (Zujić Atalić et al 2014). Two years after, KPC-2 producing strains rapidly spread in northwest Croatian region causing hospital outbreaks (Bedenić et al 2015, 2016). Southern regions of Croatia remained spared of KPC producing isolates until 2018. The rate of KPC producing K. pneumoniae in UHC Split, the largest hospital centre on eastern Adriatic coast approaches nowadays 12%, presenting a major national concern. In Europe, particularly high prevalence of KPC carbapenemases in K. pneumoniae ST258, associated with hospital outbreaks, was reported in two Mediterranean countries—Italy and Greece (reported to have an “endemic situation”), as well as Poland (Canton et al. 2012; Giani et al 2013; Van Duin and Doi 2017). Outbreak isolates in Italy were also found to be colistin resistant limiting the therapeutic options (Mammina et al. 2012). The hospital isolates from three centers exhibited similar resistance patterns except that Dubrovnik isolates demonstrated frequent colistin resistance. The transonjugants strains were shown to be positive for blaKPC genes and harboured FIIs plasmid, indicating the plasmid location of the carbapenemase encoding gene. Unlike L plasmid carrying blaOXA-48 in the previous study (Bedenić et al 2018), FIIs plasmid harbouring blaKPC in this study was highly transferable. This is in concordance with the previous studies which found KPC carbapenemases encoded on highly transferable FIIs plasmid (Baraniak et al 2011). Cefotaxime resistance was not transferable even in CTX-M producing organisms which could be explained by chromosomal incorporation of blaCTX-M genes, as reported in the previous study (Avgoulea et al 2018). Despite high level cefoxitin resistance, pAmpC were not found. None of the isolates was defined as hyperviscous which is in contrast with the previous studies which found correlation between hyperviscousity and KPC production (Vargas et al 2019).
This study demonstrated the ability of K. pneumoniae to accumulate different resistance determinants, including carbapenemases, ESBL, and colistin resistance determinants. Two different reservoirs of KPC-producing K. pneumoniae were identified: one disseminating in the hospital setting and the other found in the long-term care facilities. The majority of isolates reported in this study were resistant to gentamicin in contrast to the KPC-producing isolates described in early stage of dissemination which were uniformly susceptible to gentamicin . Furthermore, in this study, KPC was the sole carbapenemase in all tested isolates, unlike the previous publication which identified three different carbapenemases in one isolate, including VIM-1, NDM-1 and OXA-48 . In the previous studies, urinary tract infections were associated with ESBLs, mainly belonging to CTX-M family (Krilanović et al 2020), but carbapenemases were reported also among urinary isolates of Enterobacteriaceae (Petrosillo et al 2015) limiting therapeutic options.
All isolates showed resistance to serum bactericidal activity and half of them hypermucosity. These virulence factors could increase the ability of the isolates to cause severe forms of UTI such as pyelonephritis or bloodstream infections.
The strength of the study is the detailed molecular analysis of the isolates from three different regions in Croatia, but the limitation is the small number of isolates per centre. Although there are many publications on the prevalence of carbapenemases in Europe, the studies analyzing the detailed genetic background of the carbapenem-resistance in urinary tract isolates of K. pneumoniae are still scarce in scientific bibliography.
Sulphamethoxazole/trimethoprim, and colistin could still be regarded as potential therapeutic options, but only ceftazidime/avibactam remains with no resistance described so far in KPC-producing isolates from this part of the Mediterranean basin. High proportions of KPC-producing isolates together with limited treatment options even in urinary tract infections, pose a great challenge in clinical practice all over the world. Control measures and rational antibiotic usage could have a potential role in containing the further spread of carbapenemase-producing isolates.
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The authors do not declare any conflict of interest.
The Ethical permission was not necessary. The study did not involve human or animal subjects. It is in vitro study. Urine samples were collected for purpose of routine diagnostic and informed consent was not necessary.
The part of the results is in shown in European Congress for Clinical Microbiology and Infectious Diseases (ECCMID) 2020 online library (the congress was cancelled).
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Communicated by Erko Stackebrandt.
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Bedenić, B., Sardelić, S., Bogdanić, M. et al. Klebsiella pneumoniae carbapenemase (KPC) in urinary infection isolates. Arch Microbiol (2021). https://doi.org/10.1007/s00203-020-02161-x
- Klebsiella pneumoniae
- Urinary tract infections