Enhanced survival of ST-11 carbapenem-resistant Klebsiella pneumoniae in the intensive care unit

To the Editor—Klebsiella pneumoniae, a gram-negative bacterium of the Enterobacteriaceae, is a well-known and major pathogen. Carbapenems are the mainstream agent of choice against K. pneumoniae–producing extended-spectrum β-lactamases (ESBLs). However, carbapenem-resistant K. pneumoniae (CRKP) has increased rapidly worldwide and has become an urgent threat to public health. CRKP belongs to various sequence types (STs), and ST-11 is the dominant type of CRKP in China.¹ However, the factors contributing to the dominance of ST-11 remain largely unknown. Admission to the intensive care unit (ICU) and prolonged length of stay in the ICU are major risk factors for CRKP infection.^2–4 We hypothesized that ST-11 may have better survival in the ICU than other types of CRKP. We investigated the survival of ST-11 CRKP, a few other types of CRKP, and carbapenem-susceptible K. pneumoniae (CSKP) on a polyvinyl chloride (PVC) surface at the room temperature and humidity in a real ICU situation.

We selected 6 ST11 CRKP strains, 4 CRKP of other STs (ST1, ST15, ST37, and ST45) and 1 CSKP strain (ST1229) for study. Bacteria from frozen stocks were cultured on Luria-Bertani (LB) agar overnight at 37°C. A single colony of each strain was incubated in 5 mL LB broth for ~3–5 hours to midexponential growth phase; the mixture was then centrifuged, washed, resuspended in 1 mL PBS, and finally adjusted to 0.5 MacFarland turbidity-equivalent standard (10⁸ CFU/mL). Aliquots (10 μL) of each strain were added to wells (3 wells per strain) of a sterile PVC 96-well plate (Corning, NY). The plates were dried in ambient air overnight in a clean biosafety cabinet. Afterward, the plates were put into a paper box, which was placed in a corner of the corridor in an ICU ward of our hospital. This location was away from patient-care areas, and ICU staff were notified of the location to avoid any potential contact. The temperature and relative humidity of the ICU were maintained at 25 ± 2°C and 54 ± 4%, respectively. To recovery bacterial cells, 200 μL PBS was added into each well and shaken at 85 ± 5 rpm for 60 minutes in a miniature orbital shaker (BETS-M1; Qilinbeier, Haimen, Jiangsu, China). The 100-μL bacterial suspension was streaked on LB agar, which was incubated at 37°C overnight, and the number of colonies was counted manually. The biofilm formation of these strains was facilitated by aerobic incubation at 37°C in a microtiter plate system, as described previously.⁵ All statistics were determined using GraphPad Prism version 8.0.2 software (GraphPad Software, San Diego, CA). The statistical techniques employed for analysis were descriptive statistics and unpaired t test with a Welch correction.⁵P values < .05 were considered statistically significant.

All samples survived for >3 days on the PVC surface in the ICU (Table 1). ST-11 strains exhibited longer survival than non–ST-11 strains (P = .024) (Fig. 1, panel A). No significant difference was observed in biofilm formation between ST-11 and non–ST-11 strains in this study (P = .235), suggesting that the prolonged survival of ST11 strains was not due to biofilm formation (Fig. 1, panel B). A previous study also found no relationship between biofilm formation of K. pneumoniae and the transmission in ICU.⁶

Table 1. Strains and Their Survivals in the Intensive Care Unit

Note. ST, sequence type; OD, optical density; ICU, intensive care unit; RICU, respiratory ICU.

Fig. 1. Survival and biofilm formation of ST11 and non-ST11 K. pneumoniae. Panel A, duration of survival days of ST11 and non-ST11 K. pneumoniae. Panel B, biofilm formation of ST11 and non-ST11 K. pneumoniae.

Therefore, ST-11 CRKP shows an advantage in survival days compared to non–ST-11 CRKP, which may contribute to its wide spread in healthcare settings such as the ICU. To our knowledge, this is the first study to demonstrate that ST-11 K. pneumoniae strains can survive longer than several other types of CRKP in a real ICU environment. These findings support the essential role of environmental cleaning in preventing the in-hospital transmission of CRKP.