Aim: The aim of the current study is to determine: (1) the prevalence of extended-spectrum β -lactamase-producing K. pneumoniae (ESBL-Kp) isolated from clinical samples and a hospital environment in Hassan II Hospital (Settat, Morocco); (2) the associated risk factors of ESBL-Kp infections; (3) the link between clinical and environmental isolates. Methods: During the study period (April 2010 to March 2011), all patients infected and hospital environment sites contaminated by K. pneumoniae were considered as the potential study population and environmental site. The clinical data were collected to identify risk factors for ESBL carriage of K. pneumoniae infection. Screening of ESBL-and carbapenemase-producing isolates was performed by using a double-disk synergy test and the modified Hodge test, respectively. ESBL-Kp isolates were tested for the presence of genes encoding β-lactamases and were investigated by PCR. The clonal relationship between ESBL-producing isolates was analysed by ERIC- and REP-PCR method. Results: The overall prevalence of ESBL-Kp among clinical and environmental K. pneumoniae isolates was 35.13% (13/37) and 4.04% (4/99), respectively. The main risk factors for carrying ESBL-Kp were renal disease (46.15%), recent surgery (53.84%), previous hospitalisation (76.92%), and the presence of many invasive devices (53.84%). All ESBL isolates were multidrug resistant. The bla CTX-M group1and bla SHV (70.58% for each) were the most prevalent followed by bla TEM (52.94%). Thirteen strains expressed at least two bla genes. One isolate was positive in the modified Hodge test and was a bla OXA-48 producer. ERIC and Rep-PCR methods revealed an epidemic clonal dissemination of these isolates. Conclusion: The emergence of OXA-48 carbapenemase, endemic clonal dissemination and multi-drug resistance of ESBL-Kp isolates in our institution is highly alarming.
Klebsiella pneumoniae is an important pathogen known to cause nosocomial infections, such as urinary tract as well as respiratory tract infections and septicemia. Moreover, the infections caused by this isolate can be extended by the hospital environment. K. pneumoniae persists to be the major ESBL-producing organism isolated in the hospital settings all over the world. Infections due to ESBL-producing K. pneumoniae (ESBL-Kp) increased mortality in hospitalised patients [
The present study aims to determine the rate of ESBL-Kp isolated from clinical samples and hospital environment sites, and to find the relation between them. We have also determined the resistance of ESBL strains to other antimicrobial groups and analysed risk factors associated with the rate of infections by ESBL-producing strains.
Patients’ recruitment took place between April 2010 and March 2011 at Hassan II Hospital. This hospital is situated in Settat city (72 km on the north side of Atlantic Casablanca, Morocco). The hospital structure is composed of five major departments (emergency and intensive care, mother child, surgery, medicine and medico- technical) with a capacity of 302 beds. The Hassan II Hospital is a referral hospital for a population of approximately 1 million in the Settat area.
In this study, patients admitted to Hassan II Hospital were required to have nosocomial K. pneumoniae isolates coming from clinical specimens during their hospital stay. According to the Center for Disease Control (CDC) criteria, the diagnosis of nosocomial infection was established. Previous hospitalisation was defined as an admission at Hassan II Hospital or another hospital within 30 days prior to the current admission. The origin of the isolate was accepted as nosocomial if the strain was isolated more than 48 hours after hospitalisation.
Microbiological specimens were collected when the attending physician suspected infection based on systemic signs (unexplained fever, chills and hypotension) and/or local signs (purulent tracheal aspirates in mechanically ventilated patients, purulent urinary drainage, or pus or pain at a vascular catheter insertion site). Microbiological specimens were collected as recommended by the CDC.
Data involve basic demographic characteristics (age, sex, pre-infection hospital stay, and nosocomial origin), in addition to the comorbid diseases (surgical intervention; renal, respiratory and central nervous diseases; along with many more). The data involve also intensive care unit admission, previous hospitalisation, recent surgery, and duration of hospital stay.
Environment samples were collected from the equipment and surfaces of the medical, intensive care unit (ICU), surgery, paediatric and maternity wards. Sampling of contaminated surface can be done with cotton-tipped swabs (Copan Diagnostics, California, USA), moistened with nutrient broth before use then placed in 1.5 mL of the same solution. Samples were then vortexed before being incubated overnight at 35˚C.
After being incubated overnight, the nutrient broth samples were recorded to show either growth or no growth, based on the presence of a turbid solution. The samples showing growth were then plated onto Mac Conkey (MAC) agar and incubated overnight at 35˚C. Any colonies on MAC agar were identified using conventional methods, as well as the commercial identification kit API 20E (Biomerieux, Marcy l’Etoile, France).
Antimicrobial drug susceptibility testing for K. pneumoniae isolates was performed according to the disk diffusion method on Mueller-Hinton agar medium, and the results were interpreted according to the recommendations by the Clinical and Laboratory Standards Institute (CLSI) [
The detection of ESBL among AmpC hyper-producers was assessed by a double combination disk test on cloxacillin (250 µg/mL) agar [
Total DNA was extracted by the boiling method by suspending a few colonies of overnight culture of K. pneumoniae isolates growing on Luria Bertani agar (Bio-Rad) in 500 μL of DNase-and RNase-free water (Invitrogen, Paisley, UK). The suspension was boiled at 100˚C for 10 minutes in a thermal block (Polystat 5, Bioblock Scientific, France), then centrifuged at 19,000 x g for 5 minutes. An aliquot of 1 μL of the supernatant was used as the DNA template for PCR.
K. pneumoniae isolates were screened by PCR for the following β-lactamase-encoding genes: blaCTX-M group 1, blaCTX-M group 9, blaCTX-M group 2, blaTEM, blaSHV, blaOXA-48, blaNDM, blaKPC and blaVIM as described previously [
The ERIC and REP (Enterobacterial Repetitive Intergenic Consensus and Repetitive Element Palindromic)-PCR, was performed using the primers ERIC-2 and Rep-2 [
To identify variables associated with ESBL-Kp, a risk-factor analysis was performed using a case-control study format. Demographics and hospitalisation variables of ESBL-Kp case patients were compared with patients with non-ESBL-Kp isolates. Data were entered into a database using SPSS 20.0 for Windows (SPSS Inc, Chicago, USA). The χ2 test and the independent samples t test were used for categorical and continuous variables, respectively. A stepwise multivariate logistic regression was lead to examine the association of risk factors controlling for potential confounders. The logistic model included all variables for which a p-value of < 0.1 was obtained in the multivariate analysis. A p-value of <0.05 was considered as significant.
Between April 2010 and March 2011, 37 of 192 (19.27%) infected patients were included in this study and had K. pneumoniae isolates. These patients were 62.16% female and 37.84% male (F: M ratio was 1.64).Thirteen patients presented ESBL-Kp infection (35.13%) and 24 patients had non-ESBL-Kp infections. The main epidemiological data of the infected patients by ESBL-Kp and non-ESBL-Kp isolates are shown in
The statistical analysis showed that there was no significant difference between patients infected by ESBL-Kp and non-ESBL-Kp isolates in the infected regions. In both patients groups, the most common site of infections was the urinary tract, which yielded growth mostly in patients infected by ESBL-Kp isolates (
Several factors, such as a previous hospitalisation, renal diseases, admission to surgical ward and urinary tract infection, were found to be significantly associated with an increased risk for acquisition of ESBL-Kp isolates (
A total of 1000 samples were collected during the study period. Out of these cultured samples, the growth of at least one bacterial type was observed in 26.5% (n = 265), of which 115 were from equipment (monitor, aspirator, ventilator, laryngoscope, etc.) and 150 were from the care cart (n = 60), mattress cover (n = 40) and washbasin (n = 50).
K. pneumoniae strains were identified in 99 (37.35%) cases, and according to the contaminated ward, the medical ward (52%) was heavily contaminated followed by the ICU, paediatric, surgery and maternity wards (
Variable | All isolates n = 37(%) | ESBL-Kp isolates n = 13 (%) | Non-ESBL-Kpisolates n = 24 (%) | p-value |
---|---|---|---|---|
Demographics | ||||
Age in years (mean ± SD) | 43 ± 20 | 54.53 ± 21.36 | 37.6 ± 17.6 | 0.0265 |
Gender | ||||
Female | 23 (62.16) | 4 (30.76) | 19 (79.13) | 0.005 |
Male | 14 (37.83) | 9 (69.24) | 5 (20.87) | 0.003 |
Sex-ratio | 1.62 | 0.45 | 3.8 | |
Population | ||||
Adult | 19 (51.35) | 4 (30.76) | 15 (62.5) | NS |
Elderly | 17 (45.94) | 8 (61.53) | 9 (37.5) | NS |
Child | 1 (2.70) | 1 (0.6) | 0 | NS |
Previous hospitalisation | 19 (51.35) | 10 (76.92) | 9 (37.5) | 0.02 |
Duration of hospitalisation (mean ± SD) | 11.32 ± 13.03 | 15.30 ± 14.23 | 9.17 ± 12.09 | NS |
Hospital admission ward | ||||
Surgical | 9 (24.32) | 7 (53.84) | 2 (8.33) | 0.004 |
Maternity | 14 (37.83) | 0 | 14 (58.33) | 0.0003 |
Medical | 6 (16.21) | 2 (15.38) | 4 (16.66) | NS |
ICU | 8 (21.62) | 4 (30.76) | 4 (16.66) | NS |
Infected regions | ||||
Respiratory samples | 4 (10.81) | 2 (15.38) | 2 (8.33) | NS |
Urine samples | 22 (59.45) | 9 (69.23) | 13 (54.16) | NS |
Blood samples | 2 (5.40) | 1 (7.69) | 1 (4.16) | NS |
Vaginal samples | 5 (13.51) | 0 | 5 (20.83) | NS |
Others | 4 (10.81) | 1 (0.6) | 3 (12.5) | NS |
Comorbidities | 20 (54.05) | 12/13 (92.30) | 8/24 (33.33) | NS |
Diabetes mellitus | 5 (13.51) | 0 | 5 (20.83) | NS |
Renal disease | 6 (16.21) | 6 (46.15) | 0 | 0.0007 |
Anaemia | 1 (2.70) | 1 (0.6) | 0 | NS |
Cerebro-vascular accident | 1 (2.70) | 1 (0.6) | 0 | NS |
Status epilepticus | 3 (8.10) | 3 (23.07) | 0 | NS |
Others | 5 (13.51) | 2 (15.38) | 3 (12.5) | NS |
Reason for hospitalisation | ||||
Childbirth | 11 (29.72) | 0 | 11 (45.83) | 0.003 |
Urinary tract infection | 7 (18.91) | 3 (23.07) | 4 (16.66) | NS |
Surgery | 11 (29.72) | 7 (53.84) | 4 (16.66) | 0.027 |
Bronchopulmonary infections | 3 (8.10) | 2 (15.38) | 1 (4.16) | NS |
Polytrauma | 3 (8.10) | 1 (7.70) | 2 (4.16) | NS |
Toxaemia | 2 (5.40) | 0 | 2 (8.33) | NS |
Type of invasive device | ||||
Intubation + ventilation | 5 (13.51) | 1 (7.70) | 4 (16.66) | NS |
Central venous catheter | 25 (67.56) | 5 (38.46) | 20 (83.34) | 0.005 |
Intubation + urinary catheter+ ventilation | 3 (8.10) | 3 (23.07) | 0 | 0.03 |
Urinary catheter+ central venous catheter | 4 (10.81) | 4 (30.76) | 0 | 0.01 |
NS: Not significant.
Proportion of positive cultures (%) | Proportion of samples with K. pneumoniae present (%) | |
---|---|---|
Sample type | ||
Surface | (150/265) 56.6 | (60/99) 60.6 |
Equipment | (115/265) 43.4 | (39/99) 39.4 |
Wards | ||
ICU | (60/200) 30 | (28/60) 46.66 |
Medical | (50/200) 25 | (26/50) 52 |
Maternity | (40/200) 20 | (10/40) 25 |
Paediatric | (45/200) 22.5 | (15/45) 33.34 |
Surgery | (70/200) 35 | (20/70) 28.57 |
Total | (265/1000) 26.5 | (99/265) 37.35 |
The antimicrobial susceptibility testing was done by the disk diffusion method of clinical and environmental isolates of K. pneumoniae.
Among 37 clinical isolates, the resistant rates for the β-lactam antibiotics amoxicillin/clavulanic acid, cephalothin, third-generation cephalosporins (ceftriaxone, ceftazidime, cefotaxime), aztreonam and cefoxitin were 81.08%, 81.08%, 35.14%, 35.14% and 16.22%, respectively. The resistance rates for the non-β-lactam antibiotics cotrimoxazole, ciprofloxacin and gentamicin were 48.65%, 45.95% and 32.43%, respectively.
Among the environmental K. pneumoniae isolates, 40.40% were resistant to amoxicillin/clavulanic acid, cep- halothin and ciprofloxacin. Resistance rates to other antibiotics were as follows: 38.38% to cotrimoxazole, 28.28% to gentamicin, 13.13% to cefoxitin and 4.04% to the third-generation cephalosporins. Of the 17 ESBL-Kp isolates, including environmental and clinical isolates, all of them were multidrug-resistant and we noted that the resistance level was significantly higher than non-ESBL-Kp isolates (
The results of ESBL-encoding gene detection by PCR revealed that the strains studied harboured a diversity of β-lactamases, namely SHV, CTX-M and TEM (
ERIC and REP-PCR methods were used to subtype 12 clinical and environmental ESBL-Kp isolates from during the first quarter of 2011(
Antimicrobial agents | Resistance ratios, % (no. resistant strains) | |||||||
---|---|---|---|---|---|---|---|---|
Clinical strains | p-value | Environmental strains | p-value | |||||
All | ESBL (n = 13) | Non-ESBL (n = 24) | All | ESBL (n = 4) | Non-ESBL (n = 95) | |||
Amoxicillin/clavulanic acid | 81.08 (30) | 100 (13) | 70.83 (17) | 0.03 | 40.40 (40) | 100 (4) | 37.89 (36) | 0.02 |
Ciprofloxacin | 45.95 (17) | 92.3 (12) | 20.83 (5) | <0.0001 | 40.40 (40) | 100 (4) | 37.89 (36) | 0.02 |
Ceftriaxon | 35.14 (13) | 100 (13) | 0 | <0.0001 | 4.04 (4) | 100 (4) | 0 | <0.0001 |
Ceftazidime | 35.14 (13) | 100 (13) | 0 | <0.0001 | 4.04 (4) | 100 (4) | 0 | <0.0001 |
Cefotaxime | 35.14 (13) | 100 (13) | 0 | <0.0001 | 4.04 (4) | 100 (4) | 0 | <0.0001 |
Cephalothin | 81.08 (30) | 100 (13) | 70.83 (17) | 0.03 | 40.40 (40) | 100 (4) | 37.89 (36) | 0.02 |
Cefoxitin | 16.22 (6) | 38.5 (5) | 4.16 (1) | 0.01 | 13.13 (13) | 25 (1) | 12.63 (12) | NS |
Imipenem | 0 | 0 | 0 | - | 0 | 0 | 0 | - |
Ertapenem | 0 | 0 | 0 | - | 1.01 (1) | 25 (1) | 0 | 0.04 |
Aztreonam | 35.14 (13) | 100 (13) | 0 | <0.0001 | 0 | 0 | 0 | - |
Amikacin | 0 | 0 | 0 | - | 0 | 0 | 0 | - |
Gentamicin | 32.43 (12) | 84.6 (11) | 4.16 (1) | <0.0001 | 32.43 (28) | 100 (4) | 25.26 (24) | 0.005 |
Cotrimoxazole | 48.65 (18) | 61.5 (8) | 41.66 (10) | NS | 48.65 (38) | 50 (2) | 37.89 (36) | NS |
NS: Not significant.
Code | Date of isolation | Sex/age | Source | Ward | β-lactamase type | Genotype of ESBL producer | ||
---|---|---|---|---|---|---|---|---|
ERIC-PCR* profile | REP-PCR* profile | Clone | ||||||
Kp2 | 24/01/2011 | M/70 | Urine | Surgical | CTX-M, SHV, TEM | I | 1 | CK1 |
Kp4 | 01/02/2011 | - | Mattress cover | Surgical | CTX-M, TEM | CK1 | ||
Kp6 | 18/01/2011 | M/36 | Urine | Surgical | CTX-M, SHV | II | 2 | CK2 |
Kp7 | 01/02/2011 | - | Care cart | Surgical | CTX-M, SHV | CK2 | ||
Kp8 | 01/02/2011 | M/70 | Urine | Surgical | CTX-M | CK2 | ||
Kp10 | 01/02/2011 | - | Care cart | Surgical | CTX-M, SHV | CK2 | ||
Kp12 | 05/01/2011 | M/58 | Urine | Surgical | CTX-M, SHV | CK2 | ||
Kp13 | 04/03/2011 | M/64 | Pus | Surgical | SHV | CK2 | ||
Kp14 | 03/03/2011 | - | Washbasin | Surgical | CTX-M,SHV,TEM,OXA-48 | III | 3 | CK3 |
Kp15 | 09/03/2011 | F/21 | Urine | ICU | CTX-M, SHV, TEM | CK3 | ||
Kp16 | 09/03/2011 | F/21 | Protected distal bronchial | ICU | SHV, TEM | VI | 4 | CK4 |
Kp17 | 09/03/2011 | F/21 | Protected distal bronchial | ICU | SHV, TEM | CK4 | ||
Kp1 | 28/09/2010 | M/70 | Urine | Surgical | CTX-M, TEM | ND | ND | |
Kp3 | 24/04/2010 | M/71 | Urine | Medical | CTX-M, TEM | ND | ND | |
Kp5 | 13/07/2010 | M/65 | Blood | ICU | CTX-M, TEM | ND | ND | |
Kp9 | 13/04/2010 | F/74 | Urine | Medical | SHV | ND | ND | |
Kp11 | 06/10/2010 | M/68 | Urine | Medical | SHV | ND | ND |
ND: Not determined; * The ERIC-PCR and REP-PCR profiles for K. pneumoniae isolates are shown in
represented by two strains, one isolated in the surgical ward from the environment (washbasin) and the other in ICU from a hospitalised patient. The clone CK4 presented the same band between two clinical strains isolated from the ICU.
During the last few years, ESBL-producing K. pneumoniae represented the main cause of severe infections, not only in hospitals but also in community settings. The presence of ESBL restrains the activity of a wide number of antibiotics, leading to serious therapeutic difficulties with an important impact on the outcome of the patients [
The length of hospital stay mainly in ICU, mechanical ventilation, urinary catheterization, arterial catheterization, the use of a central venous catheter, and previous antibiotic treatment were found to be risk factors for the acquisition of ESBL-Kp infection along with many more [
The ERIC- and REP-PCR methods revealed four different profiles among the 12 ESBL-Kp isolates tested. The unit that was the most affected by the presence of ESBL-Kp isolates was surgery and its environmental sites (three clones). The spread of ESBL producers from one patient to another using equipment of the hospital environment has been reported many times [
In general, the resistance patterns of ESBL-Kp studied here were alike to those commonly described in other studies, the ESBL producers were resistant to different antibiotic families, including fluoroquinolones, aminoglycosides and trimethoprim/sulfamethoxazole [
In this study, one ESBL-Kp isolate was resistant to ertapenem, and was found to carry the blaOXA-48 gene; the latter was the most frequent gene reported in North African and Mediterranean countries [
The screening of blagenes detected a diversity of β-lactamases, and the CTX-M type was the most common ESBL in our hospital. The predominance of CTX-M-15 is now common in Morocco, as in other countries, as a result of worldwide expansion [
In conclusion, several clones of ESBL-Kp are widespread and are circulating both in the hospital environment and in patients. These strains harbour two or more β-lactamases genes along with the emergence of blaOXA-48 gene. A variety of risk factors associated with the occurrence of ESBL-Kp were detected. In summary, there is an urgent need to increase hygiene measures in order to limit the spread of these multidrug-resistant organisms in the hospital.
SamiraNatoubi,AbouddihajBarguigua,Sanaa BouhaliZriouil,NezhaBaghdad,MohammedTiminouni,AbderraoufHilali,SouadAmghar,KhalidZerouali, (2016) Incidence of Extended-Spectrum Be-ta-Lactamase-Producing Klebsiella pneumoniae among Patients and in the Environment of Hassan II Hospital, Settat, Morocco. Advances in Microbiology,06,152-161. doi: 10.4236/aim.2016.63015