The High Incidence of Carbapenem-Resistant Klebsiella pneumoniae in Urine from Elderly Hospital Patients May Facilitate the Spread of Resistant Strains to the Community

Abstract

Almost all European countries are affected by the expansion of carbapenem-resistant Enterobacteriaceae occurring during recent years. In the two hospitals of Verona, Italy, the incidence of carbapenem-nonsusceptible Klebsiella pneumoniae (CNSKP) began to increase by the first months of 2011, reached a peak in the summer of the same year, and currently is around 30%. Contrary to what was reported by other hospitals and although significant percentages of CNSKP were detected in respiratory samples, blood and pus, urine from hospitalized patients, mainly geriatrics, are the clinical samples with the highest incidence of these strains. Elder patients are frequently transferred from the hospital to their own homes or long-term care facilities and vice-versa. Moreover, urinary tract infections are not considered as a severe pathology and frequently is asymptomatic in elderly. For these reasons, the presence of carbapenem non-susceptible bacteria in the urinary tract of geriatric patients might be an underestimated cause of multiresistant strain spreading to the non-hospitalized population and the community.

Introduction

The Enterobacteriaceae, most notably Escherichia coli and Klebsiella pneumoniae (KP), are among the most important causes of serious hospital-acquired and community-onset bacterial infections in humans.²⁴ In these bacterial species, of special concern is the development of resistance to the carbapenems (CR), since these agents are often the last line of effective therapy available for the treatment of infections caused by multiresistant Enterobacteriaceae.^20,27 Until recently, CR-Enterobacteriaceae were extremely rare and usually associated to a decrease of the outer membrane permeability and to overexpression of the AmpC β-lactamase. More recently, a new class of bacterial enzymes capable of inactivating carbapenems, known as carbapenemases, appeared and currently the spread of carbapenemase producers is a relevant clinical issue. Carbapenemase enzymes fall into Ambler classification in groups A, B, and D. Class A (serine carbapenemase) enzymes include enzymes such as Klebsiella pneumonia carbapenemase (KPC) and imipenemase and are commonly present in members of Enterobacteriaceae. These classes of enzymes usually hydrolyze penicillin and cephalosporins more efficiently than carbapenems and are inhibited by boronic acid and less by clavulanate. Class B enzymes are metallo-beta lactamases, including Verona integron-encoded metallo-β-lactamase and New Delhi metallo-β-lactamase among others. These occur in various genera of Enterobacteriaceae and nonfermenters and have a wide spectrum of activity against carbapenem, penicillin, and extended spectrum cephalosporins but not aztreonam. In addition, they resist hydrolyses by clavulanate and are inhibited by chelating agents such as EDTA. Class D carbapenemases belong to the OXA family (oxacillinase-48 is the most frequently detected) and are commonly present in Acinetobacter spp and occasionally in Enterobacteriaceae and Pseudomonas spp. These enzymes hydrolyze carbapenems weakly and are poorly inhibited by clavulanate.

KP carbapenemases have rapidly spread in the United States and are increasing elsewhere in the world⁸; by 2007, the proportion of carbapenem-resistant strains among all isolates of KP was 10%, and in some areas, the proportion reached as high as 30%.⁸ KPCs are Ambler molecular class-A carbapenemases that typically reside on transferable plasmids and can hydrolyze all penicillins, cephalosporins, aztreonam, and carbapenems. KP is the most common organism associated with KPC resistance determinants. However, KPCs are increasingly reported in other genera of the Enterobacteriaceae family, such as Escherichia, Proteus, Serratia, Salmonella, and Citrobacter.⁸ Moreover, KPC resistance has been reported in inherently resistant organisms such as Acinetobacter baumannii and Pseudomonas spp. The rapid global dissemination of KPC genes is challenging and can be attributed to a combination of three major social and microbiological mechanisms: international travel, patient-to-patient transmission of KPC-producing organisms, and interspecies transfer of KPC-resistant elements.^5,8,18 KPC-producing organisms can colonize and cause infections similarly to their wild-type counterparts. Many infections are either systemic infections or urinary tract infections (UTIs), particularly in immunocompromised patients.⁸ Bacteria producing KPC rarely manifest as community-onset infections in nonendemic regions without any prior healthcare contact.²⁸ Indeed, risk factors for colonization or infection with KPC-producing strains are all associated with health care, including exposure to antibiotic therapy, prolonged hospitalization, intensive care unit (ICU) stay, immunosuppression, and organ transplantation.^8,9

Almost all European countries are affected by the expansion of carbapenem-resistant Enterobacteriaceae,¹⁹ even if the epidemiological scale of the diffusion is widely variable. In 2010, Italy was one of the European countries with high incidence (15%) of these antibiotic-resistant bacterial strains. In a recent review,⁸ it is reported that Italy now has low-level KPC endemicity following the first report of KPC-3-producing KP in Tuscany in 2008.¹⁷ The same report indicates that, indeed, dissemination of KPC producers continued relentlessly in Italy.⁸ A recent study from Micronet, an Italian network of 15 hospitals, indicated a global incidence of carbapenem-nonsusceptible Klebsiella pneumoniae (CNSKP) increasing from 2% in 2009 to almost 20% in the first half of 2012.²⁶ In this report and other studies, the highest incidence of multiresistant Enterobacteriaceae was found in respiratory samples and pus and blood cultures. Carbapenem-resistant Gram-negative bacteria have been isolated also from other clinical samples such as surgical lesions, liquor, and urine.¹⁰ For a comparison with other Italian and European hospitals, in the present study, the incidence of CNSKP was evaluated in the clinical samples obtained from January 2009 to June 2012 in the Azienda Ospedaliera Universitaria Integrata (AOUI) of Verona in north-eastern Italy.

Materials and Methods

In this study, we have considered the KP strains nonsusceptible (intermediate and resistant) to ertapenem, imipenem, and/or meropenem isolated from different clinical samples, mainly bronchoaspirates, bronchoalveolar lavages, blood, pus, urine, cerebrospinal fluids, and other biological fluids. Because ertapenem is an important molecule, being the first antibiotic, among carbapenems, to which resistance develops,²⁹ the data for ertapenem resistance, in addition to resistance to the more frequently used imipenem and meropenem, were included in this study. Within the period of 30 days, only the first isolate of CNSKP was considered. Multiple isolates from the same patient collected after an interval of 1 month were, however, included. The incidence of CNSKP strains was calculated as the number of carbapenem-nonsusceptible first isolates of KP divided by the total number of first isolates of KP and expressed as a percentage.

The AOUI of Verona consists of two tertiary hospitals, the Policlinico GB Rossi and the Ospedale Civile Maggiore Borgo Trento. Both hospitals are served by a single Diagnostic Microbiology Unit.

Isolates were identified at the species level and antimicrobial susceptibilities were determined using the automated system Vitek 2 System (bioMèrieux Vitek). Susceptibility to ertapenem, imipenem, and meropenem was verified using the E-test (AB Biodisk).

For the detection and monitoring of colonized patients in the hospitals from AOUI Verona, samples were inoculated in an Enterobacteriaceae selective medium (McConkey agar) with the addition of an ertapenem disk.^4,16 For high risk patients (i.e., exposed to healthcare practices or admitted in wards with patients harboring CNSKP), pharyngeal and rectal swabs were obtained once a week. This enables the clinician to activate the isolation procedure for suspected/positive patients and to decide the isolation period.

The production of carbapenemases by the CNSKP strains was tested by using the carbapenemase Nordmann-Poirel test²³ consisting on the inoculation of a carbapenem-resistant bacterial suspension in a 1-ml solution made of 3 mg of imipenem monohydrate, pH 7.8, phenol red solution and 0.1 mM ZnSO₄. The mixture was incubated at 37°C for 2 hours: a change in color from red to yellow indicates the production of carbapenemase.

The presence of bla(KPC) was investigated by polymerase chain reaction (PCR) using the following primers: KPC-fwd: TGT CAC TGT ATC GCC GTC TAG and KPC-rev: TTACTG CCC GTT GAC GCC CAA TCC.

PCR products were sequenced at Eurofins MVG Operon (Ebersberg) and compared with available sequences in the GeneBank database.

Strain genotyping was performed by multilocus sequence typing to determine the sequence type of the isolate and to establish a comparison with previously reported KPC-producing isolates. Allelic numbers were obtained on the basis of sequences of seven housekeeping genes at the www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.¹²

As in most European laboratories, from 2009 to March 2011, the Clinical and Laboratory Standards Institute (CLSI) breakpoints¹¹ were used for the evaluation of susceptibility or nonsusceptibility of KP to carbapenems, while since April 2011, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines¹⁵ were applied to perform the same evaluation. For a higher homogeneity in data interpretation, a retrospective revision and evaluation of nonsusceptibility to the three carbapenems of the strains isolated in 2009, 2010, and the first months of 2011 were conducted by using the EUCAST breakpoints. No statistically significant differences in the incidence of CNSKP were revealed in that, only four strains (out of a total of 2,000 KP isolates) considered susceptible within the CLSI guidelines resulted, in fact, as non-susceptible when the EUCAST guidelines were applied. This similarity was due to the fact that the carbapenem nonsusceptible strains showed a level of resistance with minimal inhibitory concentrations (MICs) higher than 4–8 μg/ml.

For the statistical analysis of data, the Chi-square test was used to evaluate the differences between the groups (software STATA 11).

Results

Incidence of CNSKP strains in the AOUI-Verona hospital wards

All the AOUI wards where carbapenem-resistant bacteria were isolated, were grouped into three areas, ICU, Medicine, and Surgery. During the period 2009–2012, the global incidence of CNSKP in each one of the three areas was 18%, 24%, and 14%, respectively. A significant proportion of the patients colonized/infected with CNSKP was geriatric patients and were included in the Medicine area. The global average age of patients in the AOUI-Verona is 73 years (range 19–97).

Table 1 shows the total and CNSKP strains isolated from the different hospital areas during each of the 4 years considered. The increase in CNSKP incidence began, in both hospitals, during the first months of 2011 and reached the maximum incidence in the summer of 2011 (Table 1). Whereas in the wards belonging to Medicine and ICU areas, a decrease or stabilization of the incidence was observed following the peak of maximum incidence, the percentages of nonsusceptible strains significantly increased during the same period when the Surgery area was considered (from 23 to 35%).

Table 1.

Klebsiella pneumoniae Strains Tested for Susceptibility to Carbapenems, Grouped by Type of Hospital Department from Which Clinical Samples of Any Kind Were Obtained, in the Two Hospitals in Verona, from 1 January 2009 to 30 June 2012 (n=2,267)

	2009			2010			2011			2012
Hospital department	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)
Intensive care unit	77	5	6.5% (2.1–14.5)	75	2	2.7% (0.3–9.3)	101	30	29.7% (21.0–39.6)	65	20	30.8% (19.9–43.4)
Medicine	159	1	0.6% (0.0–3.4)	219	2	0.9% (0.1–3.3)	342	148	43.3% (38.0–48.7)	161	60	37.3% (29.8–45.2)
Surgery	127	4	3.1% (0.9–7.9)	122	7	5.7% (2.3–11.5)	153	35	22.9% (16.5–30.3)	57	20	35.1% (22.9–48.9)
Other	164	0	0.0% (0.0–2.2)	176	0	0.0% (0.0–2.1)	172	15	8.7% (5.0–14.0)	97	7	7.2% (2.9–14.3)
Total	527	10	1.9% (0.9–3.5)	592	11	1.9% (0.9–3.3)	768	228	29.7% (26.5–33.1)	380	107	28.2% (23.7–33.0)

NS, nonsusceptible.

When the incidence of CNSKP was calculated per patient day, as in Fig. 1, it is possible to observe that during the period from October 2011 to April 2012, the percentage of nonsusceptible strains slowly declined. However, since April 2012, a new peak of incidence has been revealed, and in the current period of time, autumn 2012 (data not shown), the incidence in these three hospital areas is still around one third of the KP strains resulting resistant to carbapenems (34%). The epidemiological trend observed at the AOUI-Verona is similar to that presented for one of the hospitals included in the Italian Micronet network (Hospital H).²⁶

FIG. 1.

Incidence of patients colonized/infected by carbapenem-nonsusceptible Klebsiella pneumoniae at the Azienda Ospedaliera Universitaria Integrata (AOUI) in Verona from January 2010 to June 2012 from any sample collected for clinical purposes.

Incidence of CNSKP strains in clinical samples from the Verona hospitals

Table 2 shows the clinical samples where the highest incidence of carbapenem nonsusceptible strains was found. The highest incidence of CNSKP during the period 2009–2012 was detected in urine (22%), followed by respiratory samples (19%), pus and blood (17%). Comparing these data to those provided by Micronet for other Italian hospitals, it is possible to note that in the AOUI-Verona, urine samples are the clinical samples most frequently containing CNSKP, contrary to the low incidence revealed by Micronet (22% vs. 7% in the period 2009–2012). The differences in the incidence of CNSKP in the various clinical samples are more evident when the last 18 months (January 2011–June 2012) are considered: urine (40%), respiratory samples (34%), pus (30%), and blood (29%) (Table 2).

Table 2.

Klebsiella pneumoniae Strains Tested for Susceptibility to Carbapenems, Grouped by Type of Most Significant Clinical Specimen, in the Two Hospitals in Verona, from 1 January 2009 to 30 June 2012 (n=1,658)

	2009			2010			2011			2012
Clinical specimen	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)	Total number of isolates	Number NS	%NS (95% CI)
Respiratory sample	78	4	5.1% (1.4–12.6)	90	0	0.0% (0.0–4.0)	123	41	33.3% (25. 1–42.4)	63	23	36.5% (24.7–49.6)
Blood	40	1	2.5% (0.1–13.2)	57	3	5.3% (1.1–14.6)	71	20	28.2% (18.1–40.1)	43	13	30.2% (17.2–46.1)
Pus	93	5	5.4% (1.8–12.1)	84	7	8.3% (3.4–16.4)	107	24	22.4% (15.0–31.5)	45	21	46.7% (31.7–62.1)
Urine	152	0	0.0% (0.0–2.4)	185	1	0.5% (0.0–3.0)	295	128	43.4% (37.7–49.3)	132	43	32.6% (24.7–41.3)
Total	363	10	2.8% (1.3–5.0)	416	11	2.6% (1.3–4.7)	596	213	35.7% (31.9–39.7)	283	100	35.3% (29.8–41.2)

Susceptibility to other antibiotics of the CNSKP strains isolated in the AOUI-Verona

Carbapenem-resistant Enterobacteriaceae usually remain susceptible to only a few classes of antimicrobials, commonly the polymyxins, tigecycline, fosfomycin, and nitrofurantoin.¹³ There is no proven clinical efficacy against these strains and in fact there are reports of clinical failures and emerging resistance to these antimicrobials. According to an European Centre for Disease Prevention and Control (ECDC) report,¹³ in Italy, 26% of the KP strains isolated during 2010 showed multidrug resistance when fluoroquinolons, aminoglycosides, and third-generation cephalosporins were considered. When susceptibility of CNSKP strains to other antibiotics was evaluated in the AOUI-Verona, the most frequent profile was that showing susceptibility to only colistin (86.0%) and/or gentamycin (34.1%). Moreover, it was observed that 8.4% proved to be pan-resistant isolates. The MIC values for the Verona hospital CNSKP strains regarding a number of antibiotics were calculated. As for carbapenems, the MIC values varied between 2 and 32 μg/ml for imipenem and meropenem, whereas values were higher for ertapenem (32–128 μg/ml). The MIC intervals for cephalosporines such as cefotaxime and cefepime were 8 to >128 μg/ml. Most of the strains maintain their susceptibility to tigecycline (0.5–2 μg/ml) and gentamycin (1–2 μg/ml).

Molecular analyses of KP strains

All the CNSKP isolated in this study were examined for the carbapenemase production with the test described in the Materials and Methods section: 99.4% of the strains showed carbapenemase production. Of the 334 CNSKP isolated from different clinical samples at the Verona hospital, 210 strains (62%) randomly chosen from those isolated during each of the 4 years, were checked by PCR for the presence of bla_KPC. All the strains screened resulted positive for the production of the KPC-3 enzyme.

Among bla_KPC-positive KP strains, the ST258 is the major epidemic clone. This clone also spread in the Verona hospital in the considered period of time.²¹ Since the second half of 2010, after an outbreak in the Verona surgical unit,²¹ a clonal dissemination of CNSKP strains has been observed in that the KP strains identified belong to the ST512 clone, which differs in gapA locus from the ST258 clone, but is still included in the clonal complex (CC) 258.

Discussion

During the period 2007–2010, a significant increase in the incidence of carbapenem-resistant strains of KP was observed for five European countries (Austria, Cyprus, Greece, Hungary, and Italy).^2,13,16 At the end of this period, the percentage of carbapenem-resistant K. pneumoniae (CRKP) strains was 49% in Greece, 16% in Cyprus, 15% in Italy, and less than 5% in other countries. Contrary to this trend, during the years 2009 and 2010, the incidence of CNSKP was low in the two Verona hospitals (≤8%). However, starting from the first months of 2011, a high peak in incidence was observed, reaching as high as 43–46% of the strains isolated in some clinical samples showing resistance to this class of antibiotics (Table 2). During the period October 2011–April 2012, a significant decrease of CNSKP incidence occurred, but an increase was again detected coinciding with the summer period. The very restrictive measures implemented to manage and contain the resistant KP spread might have been the cause of the resistant KP lower incidence revealed, similarly to what has been reported by other authors.^1,22 The less strict application of preventive measures, for example, due to the lower availability of medical and paramedical personnel in specific periods of the year, probably renders not possible the consistency of a decreasing incidence trend.

A very significant difference in the data provided by other European and Italian hospitals and the AOUI-Verona hospitals concerns the incidence of CNSKP in urine samples. The Micronet report²⁶ concluded that the higher proportion of nonsusceptibility observed in isolates from respiratory samples, pus, and blood versus those from urine might reflect, at least in part, the out-patient origin of the urinary isolates, with out-patients being less exposed to carbapenem-resistant Enterobacteriaceae circulating in the hospital environment. On the contrary, the incidence of CNSKP strains in the Verona hospitals has been calculated in this study taken into consideration only the urine samples from hospitalized patients. It can be deduced that healthcare-associated UTIs and colonization of the urinary tract due to CNSKP are a major problem in the Verona hospitals and might constitute a concern for the containment of infections caused by CNSKP strains. This may also be the case in other hospitals whether the urine samples from hospitalized patients were analyzed. Carbapenemase-producing bacteria can colonize or infect not only those patients who are debilitated, immune-compromised, or critically ill, but also those who were previously healthy and became colonized or infected in healthcare environments practicing poor infection control.¹⁴

It is interesting to note that in 19.7% of the patients, isolation of the CNSKP strains occurs during the first 48 hours after their hospitalization. Moreover, in fifty-seven (75%) of these patients, the CNSKP strain was isolated from urine samples. This likely means that these patients arrived to the hospital already colonized/infected by carbapenem-resistant KP. Geriatric patients are, in fact, frequently transferred from the hospital to their own homes or long-term care (LTC) facilities, and vice-versa. UTI is common in LTC residents although most of these infections are asymptomatic and do not require treatment. Differentiating asymptomatic from symptomatic UTI is challenging, because LTC residents typically have chronic genitourinary complaints, multiple comorbid illnesses, and communication barriers.⁶ For elderly living in the community, UTIs represent the second most common infection. Antibiotic-resistant KP (e.g., ESBL producers) have been widely identified as community pathogens causing UTIs.³ Recent observations have shown that previous hospitalization and antibiotic treatment, as well as male gender over the age of 60 years, are risk factors for community-onset urinary tract ESBL infections.²⁵ Moreover, patients during their hospital discharge might carry carbapenem-resistant bacteria in the intestinal compartment and the urinary tract and this colonization can be associated with a high risk of developing community-onset infection. It seems plausible that patients with community-onset ESBL infections and their household contacts represent a bacterial reservoir that increases dispersal of resistance in healthy people. The extra-hospital spread of CNSKP was recently reported in a Greek study.²⁵ Similarly, infection with CRKP was associated with admission from or prior stay at a local LTC facility in West Virginia.⁷ In this report, pulsed-field gel electrophoresis analysis indicated that clinical specimens in hospitals and those from the LTC facilities were closely related.⁷

Urinary colonization/infection is not considered a severe pathology requiring, as in patients with severe infections, isolation measures. The underestimation of this concern might facilitate the spreading of resistant KP among residents in LTC facilities as well as to the nonhospitalized population and the community. Given the significant epidemiological role of KP as a cause of healthcare-associated UTI, the involvement of CNSKP in urinary infections will become increasingly common. Data regarding the incidence of CRKP in cases of urine colonization/infection from the hospitals in Verona confirm this trend. Only strict and continuous application of preventive and containment measures in the hospital, and also in domiciliary and long-term healthcare, would help to avoid diffusion of the CNSKP strains present in the urinary tract.

Footnotes

Acknowledgments

We thank Giorgio Nicolis for his help in statistical analysis of data. G. Lo Cascio and F. Soldani have equally contributed to the study.

Disclosure Statement

The authors declare that they have no conflicts of interest.

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