Extended-spectrum β-lactamases (ESBLs)-producing Gram-negative bacilli are a major cause of resistance to third-generation cephalosporins. The genes encoding these beta-lactamases are often carried in plasmids, which have allowed them to spread horizontally worldwide, and are currently endemic in community and hospital-acquired Enterobacterales. These enzymes hydrolyse third-generation cephalosporins and aztreonam, but are not active against cephamycins, and they are inhibited by clavulanic acid and by tazobactam. Currently, CTX-M, SHV and TEM family of ESBLs are the most common in most geographical areas worldwide and specifically in Spain. However, there are other less frequent families such as PER, GES or VEB.1
VEB (Vietnamese extended-spectrum β-lactamase) is a family of ESBLs, that has so far localised in class 1 integrons.2 It was first detected in Southeast Asia and subsequently in African and American countries.3,4 This bla gene has been found in Enterobacterales, Achromobacter xylosoxidans, Pseudomonas aeruginosa and Acinetobacter baumannii. In Europe it has been found in both Klebsiella pneumoniae and in non-fermentative Gram-negative bacteria since 2001.5–10 Recently, a clinical case of VEB-producing K. pneumoniae was detected in our hospital of Seville and, to date, this is the first description of VEB-1-producing K. pneumoniae in Spain.
In addition, the incidence of clinical infections caused by carbapenemase-producing organisms has increased in the last years.11 A rare plasmid-mediated variant of OXA-48 called OXA-436 has been described.12 The enzyme has been shown to be a class D carbapenemase similar to OXA-48 in terms of substrate specificity. It has a higher activity at higher temperatures, resembling a human infection scenario, whereas OXA-48 has activity at lower temperatures, indicating an environmental scenario. However, no significant difference is shown in antimicrobial susceptibility profiles in vivo.13 It was initially discovered in an Enterobacter asburiae isolate from a patient admitted to a hospital in the capital of Denmark and subsequently detected in other enterobacteria (Citrobacter freundii, K. pneumoniae, Escherichia coli) in different Danish hospitals.11,12,14 OXA-436 has also been identified in a strain of Shewanella putrefaciens from a Pakistani hospital.15
A 57-year-old woman presented to the emergency department with a one-week history of fever, chills, cough, and asthenia. She had no past medical history. She had no previous contact with health-care or rural areas, and no history of travel in the 6 months prior to admission.
Clinical examination, including gynecological examination, was unremarkable. Laboratory data revealed an increase in acute phase reactants (CRP=237mg/l, PCT=3.70ng/ml), with non-pathological urine sediment. In the computed tomography of the neck, chest and abdomen with contrast and in the cholangio-MRI no data of interest were found. Colonoscopy showed no pathological lesions.
An empirical antibiotherapy with ceftriaxone was started. Blood cultures yielded two different Gram-negative isolates: E. coli and K. pneumoniae, both identified by MALDI-TOF (Bruker).
Due to the epidemiology of our hospital, β-LACTA and β-CARBA tests (Bio-Rad) were carried out for a rapid detection of resistance. The β-LACTA test was positive in both isolates. The K. pneumoniae isolate was identified as an OXA-48 producer based on positive results in the β-CARBA test and NG-Test CARBA 5 (NG biotech). The β-CARBA test for E. coli was negative. Susceptibility was determined using the MicroScan Walk-Away (Beckman Coulter®) NMDR panel.
The K. pneumoniae isolate was susceptible to ciprofloxacin, levofloxacin, amikacin, cotrimoxazole, colistin and tigecycline, and to newer antibiotics such as ceftazidime/avibactam, meropenem/vaborbactam and cefiderocol. It was resistant to third-generation cephalosporins, amoxycillin/clavulanic acid, piperacillin/tazobactam, ceftolozane/tazobactam, ertapenem, gentamicin and tobramycin. It was susceptible with increased exposure to meropenem and imipenem, according to EUCAST breakpoints.
The patient did well clinically and was discharged after completing treatment with 12 days of ceftazidime/avibactam.
In this case, only the K. pneumoniae isolate was sent to the reference center for sequencing, since only carbapenemase-producing isolates were sent. Initial bacterial typing of OXA-48-producing K. pneumoniae was carried out by pulsed field gel electrophoresis with XbaI, and the isolate was found to be different (>6 bands of difference) to previous local isolates. Whole genome sequencing was performed by the Illumina MiSeq, using Nextera Flex, library preparation and genome assembly was performed using CLC Genomics Workbench software. Annotations of resistance determinants and MLST typing were done by using the tools from the Center for Genomic Epidemiology (CGE; https://cge.cbs.dtu.dk/services/). The isolate was assigned to clone ST37 and carried the resistance determinants blaOXA-436, blaOXA-10 and blaVEB-1. The coverage and homology of blaOXA-436 was 100% by Resfinder and CARD. It is located in a 15176 bp contig with a depth coverage of x30.5. It is identical to a blaOXA-436 sequence deposited in Genbank KY863418 of plasmidic nature, which correlates to a Danish strain of E. asburiae AMA 497.
Therefore, resistance to cephalosporins was due to the production of VEB-1 and resistance to carbapenems to the production of OXA-436 and/or OXA-10.
In Europe, the first VEB-1-producer was identified in ceftazidime-resistant P. aeruginosa isolates in Bulgaria (2001–2005).5 Subsequently, in this same species, in the United Kingdom, three more cases of VEB-1-producing P. aeruginosa were detected in 2003–2007: the first case was a patient transferred from a hospital in India; the second case was a patient transferred from the previous hospital; and the third case was a patient repatriated to the United Kingdom from Thailand.6 Subsequently, VEB-1-producing A. baumannii isolates were identified in France and in Belgium in 2003. Epidemiological investigations showed that frequent patient transfers between French hospitals and long-term care facilities in Belgium could be responsible for the spread of strains between countries. The genetic environment of blaVEB-1 revealed an integron identical that found in most P. aeruginosa isolates from Thailand.7,8
A few years later, cases of VEB-producing Enterobacteriaceae infections began to be detected in Europe. In Greece there was an outbreak of K. pneumoniae producing KPC-2 and VEB-25 (which differs from VEB-1 in only one mutation) occurred in 2019.9
In Spain, a non-conjugative VEB-4-producing isolate (this variant differs from VEB-1 in two amino acid substitutions)10 was identified in a survey of a ESBL-producing Proteus mirabilis collection in Barcelona in 2000–2005, although epidemiological data were not included in the report. Our case is the second described in our country and the first in K. pneumoniae. Most importantly, the patient had not travelled outside Spain, which could indicate that this type of enzyme has already been introduced into our country and is circulating in the community. This highlights the need for continuous monitoring of the determinants associated with third-generation cephalosporin-resistant isolates to detect changes in prevalence. Currently, there is a useful method consisting of a set of six multiplex PCRs and one simplex PCR available for rapid screening of β-lactamases, including VEB. This PCR method is a fast, low-cost and reliable tool for the screening of frequently encountered β-lactamases. It will assist in monitoring their emergence and their spread, and it could be used in epidemiological surveys.16
