Prevalence of Klebsiella pneumoniae strains producing carbapenemases and increase of resistance to colistin in an Italian teaching hospital from January 2012 To December 2014

Overall, 15,104 patients were included in the study (2,645 subjects in 2012, 5,249
in 2013 and 7,210 in 2014), and a total of 496 consecutive non-replicated strains
of CPKP were collected, as first isolate detection from each patient.

The identification was performed with the Hodge test and confirmed in 496/496 strains.
Rosco Diagnostica test was performed in a subset of 93/496 KPC/MBL isolates and we
obtained the following results : in 2012, 23 strains showed synergism with meropenem
and boronic acid, and all but one were tested and confirmed by molecular analysis;
in 2013, 64 strains showed synergism with meropenem and boronic acid (all but 12 tested
and confirmed by molecular analysis); 3 strains didn’t showed synergism and were confirmed
as OXA-48; in 2014, 3 strains showed synergism with meropenem and boronic acid and
were tested and confirmed by molecular tests. Four hundred thirty-six strains were
characterized using molecular methods: 432 KPC strains, 3 OXA-48 strains and 1 NDM
strain were identified.

Specifically, we collected 149 CPKP strains in 2012 (39 [26.2 %] from screening rectal
swabs [SRS]), 133 strains in 2013 (70 [52.6 %] from SRS) and 214 strains in 2014 (164
[76.6 %] from SRS). The number of CPKP strains isolated using SRS increased in 2013
relative to 2012 (p??0.001) and in 2014 relative to 2012 and 2013; this increase was statistically significant
(p??0.001 and p??0.001) (Fig. 1). A higher absolute percentage (relative to the number of patients) was reported
in 2014 (2.3 %), and this difference was significant relative to 2013 (1.3 %, p?=?0.0001) and 2012 (1.5 %, p?=?0.0172).

Fig. 1. Carbapenemase producing Klebsiella pneumoniae strains isolated in 2012, 2013 and 2014. CS: clinical samples. SRS: surveillance
rectal swabs

During the study period, 5,305 subjects were screened in the ICD, 7,560 were screened
in the SD, and 2,239 patients were screened in the MD. The comparative analysis of
the detection of CPKP by SRS versus CS revealed an increased rate of CPKP identified
by SRS relative to CS (Fig. 2).

Fig. 2. Carbapenemase producing Klebsiella pneumoniae strains isolated from surveillance rectal swabs and from clinical samples in 2012,
2013 and 2014. IC: intensive care. CS: clinical samples. SRS: surveillance rectal
swabs

In the ICD, the percentage of cases identified by SRS was 49 % in 2012, 75 % in 2013
and 93.3 % in 2014 (the Chi-squared test for trend, p??0.0001); a comparable figure was described in the SD, with the highest value (94.9 %)
reported in 2014, and lower percentages in 2013 (52,4 %, p??0.0001 respect to 2014) and in 2012 (23.2 %, p??0.0001 respect to 2014 and p?=?0.01 respect to 2013). The MD department was characterized by a sharp increase
between 2012 (7.3 %) and both 2013 (31.9 %, p?=?0.003) and 2014 (40.8 %, p?=?0.0001).

In the ICD, this trend increased over time (from 1.5 % in 2012 to 1.8 % in 2013, and
to 2.3 % in 2014). A statistically significant difference was demonstrated in the
SD between 2012 and 2013 (1.9 % versus 0.8 %, p?=?0.0357) and between 2013 and 2014 (0.8 % versus 2.2 % p??0.0001); in the MD the increase from 2012 (0.8 %) to 2014 (2.6 %) was significant
(p?=?0.032) (Table 1).

Table 1. Percentage of positive SRS respect to the number of the patients involved in the surveillance
in the Medical Department, Surgical Department and Intensive Care Department in 2012,
2013 and 2014

Time to first detection of SRS was analyzed by evaluating three different figures.

In 2013, 37 % of the SRS were collected as first sample within 7 days from admission
to the hospital, 45 % after a negative survey of one sample at least, 17 % after a
minimum stay of 7 days with no surveillance data available at least. Of note, 19 %
of the SRS cases were found positive after a negative survey of three samples at least.
About clinical samples, these rates were 33 %, 38 % and 29 % respectively.

In 2014, 27 % of the SRS were collected as first sample within 7 days from admission,
60 % after a negative survey of one sample at least, 13 % after 7 days at least with
no survey data. About clinical samples, these rates were 52 %, 31 % and 17 % respectively.
It has to be underlined that even more SRS cases than 2013, 31 %, were found positive
after a negative survey of three samples at least.

The MLST analysis revealed the circulation of CPKP strains previously described in
other studies, including ST-258 and ST-512. The abundance of certain prevalent types
may be partially because investigations were conducted on episodes of temporal or
spatial clusters. Nevertheless, some new ST variants were demonstrated: strains 510,
527, 868, 1081, 1207, 1326 and 1733. Interestingly, in August 2011, a new single locus
variant (SLV) of ST-512 (ST-745) was identified. Thereafter, the variant quickly spread
throughout the hospital. ST-745 was identified in 24 of 74 strains analyzed in 2011,
when 125 patients were demonstrated as infected or colonized by CPKP. MLST data obtained
in 2012, 2013 and 2014 are presented in Table 2.

Table 2. Description of the ST- identified in carbapenemase producing Klebsiella pneumoniae divided by Department in 2012 (a), 2013 (b) and 2014 (c)

Colistin-resistant strains

At the first detection in our survey, we collected a total of 399 CoS strains and
97 CoR strains (Fig. 3). The percentage of CoR cases (both SRS and CS) relative to all CPKP cases was 10.7 %
in 2012: a statistically significant increase respect to 2012 occurred in 2013 (25.6 %)
and in 2014 (22 %).

Fig. 3. Number of colistin resistant strains and colistin susceptible strains identified in
2012, 2013 and 2014. CoR: colistin resistant. CoS: colistin susceptible

The following overall distribution of MIC values was observed after screening with
Vitek: 372 strains exhibited an MIC value???0.5 mg/L, 14 strains were MIC?=?1 mg/L,
12 strains were MIC?=?2 mg/L, 9 strains were MIC?=?4 mg/L, 6 strains were MIC?=?8 mg/L,
and 83 strains were MIC???16 mg/L. A detailed description (by year) is provided in
Fig. 4. Among the strains classified as CoR at the first detection, 65 and 32 were identified
by SRS and CS, respectively (Fig. 5). No differences were observed in the CS and SRS MIC values. With respect to the
clinical samples, 12 of the 32 strains were isolated from urine cultures, 7 strains
were detected in bronchial aspirates, 6 strains from skin swabs, 2 strains from blood
cultures, 2 strains in drainage fluid, 2 strains from vaginal swabs and 1 strain from
a pharyngeal swab (Table 3). Among the 65 SRS that were found to be CoR, 38 % were collected from the patient’s
first SR. The increase proportion of CoR strains isolated at the first detection from
SRS in 2013 compared with 2012 was statistically significant (p?=?0.02) Moreover, we observed a significantly higher frequency of CoR strains detected
from SRS when we compare them respect to the number of all available CPKP cases (p?=?0.0002 in 2012 versus 2013, p?=?0.0034 in 2012 versus 2014).

Fig. 4. Colistin MIC values (mg/L) performed with Vitek® 2 system in 2012, 2013 and 2014.
MIC: minimum inhibitory concentration

Fig. 5. Colistin resistant strains at the first detection in 2012, 2013 and 2014. CS: clinical
samples. SRS: surveillance rectal samples

Table 3. Description of the first detection of a colistin resistant strain in clinical samples
in 2012, 2013 and 2014

Evolution of susceptibility to colistin

Although not from the same material (FU), a strain that was later isolated in the
same patient was also detected in 210/399 patients with a CoS stains at first detection,
allowing us to perform a longitudinal analysis. After previously identifying a CoS
strain, we identified a CoR strain in 50/210 patients (23.8 %) (Table 4). We were able to perform MLST for both the CoS and CoR strains in 17 of 50 patients
(Table 5). All pairs of isolates were obtained in 2014, with the exception of the strain from
patient 14 that was detected in 2013. The same ST was detected in both the CoS and
CoR strains in 13/18 strains. In 11/13 cases, the CoS strains was also isolated on
CS. In one case, another pathogen (Enterobacter aerogenes, susceptible only to colistin) was isolated from a CS. None of these patients were
discharged within the time interval between the two isolates.

Table 4. Longitudinal analysis of colistin susceptibility evolution in patients with a colistin
susceptible isolate at the first detection in 2012, 2013 and 2014

Table 5. MLST analysis on pairs of CoS and CoR strains obtained from the same patient. Time
to switch is intended as the time-interval between the first CoS strain isolated from
a clinical sample and the first CoR strain isolated from the same patient

Four of 18 strains exhibited a different ST between the CoS and CoR strains. Two patients
were discharged during the FU, and 2 other patients were continuously hospitalized.
Note that in one of these four cases, the CoR strain (ST1733) was a single locus variant
of the CoS strain (ST554). Patient 16 reported a simultaneous isolation of a ST258
CoS strains and of a ST512 CoR strain from the same sample at the first detection.

All cases were tested to identify the KPC type; in 15 couples both isolates were KPC-3
and in one couple both strains were KPC-2, suggesting a persisting infection with
the same strain. In one case we found a KPC-2 at baseline and a KPC-3 at follow-up:
this finding made difficult a definite interpretation.

Isolation of CPKP from blood

Seventy-eight CPKP strains were isolated from the blood of 75 patients as a first
or subsequent detection: 32 in 2012 (5 CoR), 17 in 2013 (1 CoR) and 29 (6 CoR) in
2014. The crude mortality rate among patients with CPKP isolated from the blood was
35/75 (46.7 %). In cases of poor outcomes, the median time to death was 10 days, whereas
the median time to discharge was 28 days.