Monoclonal Outbreak of VIM-1-Carbapenemase-Producing Enterobacter cloacae in Intensive Care Unit,University Hospital Centre Split,Croatia

Introduction

E nterobacter cloacae has recently emerged as an important hospital pathogen responsible for various nosocomial infections, including bacteremia, lower respiratory tract infections, skin and soft tissue infections, endocarditis, and intra-abdominal infections.^5,8,12,16,18 The most susceptible patients are those who stay in the intensive care units (ICUs) for prolonged periods. Other risk factors are serious underlying conditions, immunosuppression, use of foreign devices (such as intravenous catheters), invasive procedures (mechanical ventilation), malignancy, and previous use of antimicrobial agents. E. cloacae is intrinsically resistant to ampicillin and narrow-spectrum cephalosporins. Resistance to extended-spectrum cephalosporins and aztreonam is usually related to mutational derepression of the chromosomal Ambler class C β-lactamase or to the production of plasmid-mediated extended-spectrum β-lactamases (ESBLs). Since ESBL-producing bacteria are often multidrug resistant (MDR), carbapenems represent therapeutic agents of last resort for treating these life-threatening infections. Carbapenems have stability to hydrolysis by most β-lactamases, including ESBLs. However, the acquired metallo-β-lactamases (MBLs) have emerged among several gram-negative pathogens, Pseudomonas aeruginosa, Acinetobacter baumannii, and species of the Enterobacteriaceae family.^6,17 Among five types of acquired MBLs (VIM, IMP, SPM, GIM, and SIM-type), the VIM-type is most prevalent in Europe. ²³ ESBLs and carbapenemases are often encoded by genes located on large plasmids that also carry genes for resistance to other antimicrobial agents, such as aminoglycosides and fluoroquinolones.^11,21 Increasingly reported carbapenemases in E. cloacae clinical isolates from various geographical regions represent a matter of major concern for clinicians since this nosocomial life-threatening infections could be treated with a limited number of “last-line” agents, such as colistin.^6,10,13

Until now, and to the best of our knowledge, this is the first monoclonal outbreak of infection due to MDR VIM-1-producing E. cloacae in Croatia. The aim of this study was to describe the antimicrobial resistance, clinical background, type of β-lactamase genes, and genetic relatedness among carbapenem-resistant strains of E. cloacae isolated from patients in ICU at the University Hospital Centre Split, Croatia.

Materials and Methods

Results

In a 3-month period in 2012, five patients were infected and one patient was colonized with MDR VIM-1-producing E. cloacae strain susceptible only to colistin.

The clinical and demographic characteristics of the infected/colonized patients are summarized in Table 1. The median age was 47 years (range, 28–60 years) and five patients (83.3%) were male. All patients had serious underlying diseases: case 1 had soft palate neoplasm with metastases, two patients had complicated intracranial hemorrhage (cases 2 and 5), one patient (case 3) had complicated medical conditions, including acute respiratory distress syndrome (ARDS) and diffuse peritonitis, case 4 had polytrauma with splenic rupture and pulmonary contusion, and case 6 had multiple abscesses and sepsis with ESBL-positive Klebsiella pneumoniae. All six patients received broad-spectrum β-lactam antibiotics, including cephalosporins (cases 1, 2, 4, and 5) and β-lactam/β-lactamase inhibitor combinations (cases 3, 5, and 6). Three patients were receiving an MEM when VIM-1-producing E. cloacae was isolated (cases 2, 5, and 6). One patient was already receiving colistin when the infection occurred. All patients were on mechanical ventilation.

All isolates were considered hospital-acquired. The median time from ICU admission to the isolation of an MBL-producing strain was 16 days (range, 7–30 days). All patients had spatiotemporal epidemiological links in the ICU. Of the six resistant isolates, three were isolated from the lower respiratory tract, whereas the remaining strains were isolated from blood, abdominal cavity, and rectal swab. Five cases of clinical infection and one case of colonization with MBL-producing organism have been documented. Four (80%) of five infected patients died. Among them, three patients (case 1, 3, and 4) died before susceptibility results were available. Patient 6 failed to respond to treatment with amikacin and colistin and had a fatal outcome. Although adequate therapy is essential to treat infections caused by MBL-producing bacteria, the severity of underlying conditions is also important factor for overall outcome. Two patients have been transferred to other hospitals; patient 2 was treated with colistin and patient 5 was considered colonized without local and general signs of infection. Both patients had positive findings of MBL-producing organism at the time of hospital discharge.

All isolates showed high-level resistance to broad-spectrum cephalosporins and inhibitor-protected penicillins (piperacillin/tazobactam and ampicillin/clavulanic acid). Only one strain was ESBLs producer. All six isolates were resistant to all tested quinolones, tobramycin, netilmicin, gentamicin, and trimethoprim–sulfamethoxazole. Carbapenems were affected to different degrees (MIC, 1 to >32 mg/L). All strains were resistant to ertapenem (MIC range, 1 to >32 mg/L). Susceptibility to IMP and MEM was variable; isolate 1 was susceptible to IPM and MEM (MIC, 2 mg/L); isolate 2 was susceptible to MEM (MIC, 2 mg/L) and intermediate to IPM (MIC, 4 mg/L); isolate 3 had reduced susceptibility to MEM (MIC, 4 mg/L) and IMP (MIC, 6 mg/L); and the remaining three strains (isolates 4, 5, and 6) were resistant to MEM and IMP (MIC range, 8 to >32 mg/L). MIC values for amikacin showed intermediate susceptibility (MIC, 16 mg/L). Colistin was the only susceptible antibiotic for all six strains (MIC, ≤0.5 mg/L).

All isolates yielded positive results of the EDTA-IMP disk synergy test. PCR confirmed the presence of bla_VIM-1 gene in all clinical isolates. The characterization of six VIM-1-producing E. cloacae isolates by PFGE revealed that all of them were clonally related, showing an indistinguishable PFGE pattern (data not shown).

Discussion

Carbapenems are important therapeutic agents for the treatment of hospital-acquired infections caused by MDR gram-negative bacteria, especially those carrying genes for ESBLs and derepressed AmpC β-lactamases. The emergence and dissemination of acquired MBLs among gram-negative organisms have become a substantial global health problem and represent a significant threat to the management of nosocomial infections because of the lack of any effective antimicrobial agents. ³

However, the carbapenem resistance among Enterobacteriaceae family is still rare in Croatia. Here, we report the first detection and describe the first monoclonal outbreak of infections due to MDR VIM-1-producing E. cloacae at the University Hospital Center Split, Croatia. The emerged strain showed high-level resistance to all clinically available antimicrobial agents except to colistin. MICs of carbapenems were variable. Differences in the carbapenem resistance levels among isolates of the same PFGE type were also observed in other studies and may be due to variable expression of the MBL gene or existence of additional resistance mechanisms.^9,15,21 The mobile nature of MBL genes and their frequent association with other resistance genes make them an extremely potent threat for managing the nosocomial infections. Moreover, findings of clonally related VIM-1-producing E. cloacae isolates from different hospital wards, which persisted over time, confirm the risk of spread of these clones with the transfer of patients to different wards and their long time persistence in hospital settings. ²¹

All ICU patients at the University Hospital of Split are routinely screened for MDR bacteria on the day of admission, with use of surveillance cultures (rectal, nasal, and oropharyngeal swabs). During the study period, none of the isolated strains from basal surveillance cultures were resistant to carbapenems. Therefore, all cases were considered hospital-acquired. After the emergence of the first two ertapenem-resistant E. cloacae strains, we have implemented a multidisciplinary panel of infection control measures (reinforcement of hand hygiene measures, barrier precautions, and surveillance cultures) to prevent the further spread of the resistant clone. Among all patients who were admitted to the ICU during the study period, only one patient was colonized with an MBL-producing isolate after 21 days of hospitalization (case 5), without developing clinical infection.

The source of this monoclonal outbreak was not identified. The patient-to-patient cross-transmission via healthcare workers, overcrowding, and understaffing in the ICUs are well-known causes of the dissemination of the resistant clones. These findings highlight the limitation of therapeutic options for the treatment of infections with VIM-1-producing organisms and predict the poor clinical outcome and therapeutic failure.

The emergence of colistin resistance, which has been already documented in Enterobacteriaceae family,^1,20 was not observed in our study. Colistin is still considered the most active agent against carbapenem-resistant isolates of E. cloacae in our hospital. In conclusion, awareness, continued surveillance of carbapenem resistance, early detection of MBL-producing pathogens, and strict implementation of a multidisciplinary panel of infection control measures as well as wiser antibiotic policies have to be prioritized to limit their spread in the hospital.