Abstract: Reports on outbreak of extended-spectrum β-lactamases (ESβLs) by Enterobacteraiceae and especially Klebsiella pneumoniae, are few in Saudi Arabia. This study was therefore devoted to describe the outbreak which occurred by ESβL-producing K. pneumoniae. Sixteen K. pneumoniae isolates were isolated from 16 neonatal patients hospitalized from September 2007 to December 2007 in the neonatal intensive care unit during the outbreak in Al-Qatif Hospital, Eastern Province, Saudi Arabia. These isolates were sent to microbiological laboratories, College of Pharmacy, King Saud University, for investigation. Klebsiella pneumoniae strains were found to produce antibiotic resistance and produce extended spectrum beta-lactamase. Genotypic characterization of extended spectrum beta-lactamase producing K. pneumoniae showed that all isolates carried TEM-1, SHV-1 and CTX-M-15 genes. Matting out assay revealed that all third generation cephalosporins were located on transferable plasmid. An outbreak which occurred in neonatal intensive care unit was due to CTX-M-15-producing K. pneumoniae isolates either single or in multiple clones. This is the first report of blaCTX-M-15 gene in Saudi Arabia from K. pneumoniae and the first outbreak in Saudi hospitals due to CTX-M-15 producing K. pneumoniae.
INTRODUCTION
Plasmid mediated extended-spectrum β-lactamases (ESβLs) were first detected in a Klebsiella pneumoniae isolate in Germany in 1983 (Kliebe et al., 1985). The most common of the ESβLs are those derived from broad-spectrum β-lactamases TEM-1/-2 and SHV-1 by acquisition of specific point mutations (Nukaga et al., 2003). CTX-M-type is non-TEM and non-SHV ESβLs, which have less than 40% homology with TEM and SHV (Canton and Coque, 2006). CTX-M β-lactamases are very rapidly disseminated and are now widely dispersed geographic areas, including many parts of Europe, Asia, Africa and America (Al-Agamy et al., 2006; Canton and Coque, 2006). Epidemiological reports demonstrate that some enzymes are more frequently than others and also the predominant enzyme type varies with country and the diverse CTX-M types often exist within a single country (Livermore et al., 2007; Coque et al., 2002). Outbreaks of K. pneumoniae infections in neonates have been widely reported and are frequently associated with widespread colonization of babies, systemic infections and death (Hobson et al., 1996). Hospital outbreaks of K. pneumoniae, especially in neonatal units, are often caused by ESBL-producing strains and have been increased over the past years (Carrër et al., 2009; Randrianirina et al., 2009). No data on genotype of ESβL is available at regional level. Therefore, this study was undertaken to characterize ESβL genes in K. pneumoniae isolated from an outbreak in Neonatal Intensive Care Unit (NICU), Al-Qatif Hospital, Eastern Province, Saudi Arabia.
MATERIALS AND METHODS
Bacteria Strains
Sixteen isolates of K. pneumoniae were collected during an outbreak
recorded between the 22 September 2007 to the 4 December 2007 from NICU during
an outbreak the occurred in Al-Qatif Hospital, Eastern Province, Saudi Arabia.
These isolates were sent to the college of Pharmacy, King Saud University, Riyadh.
K. pneumoniae isolates were isolated from clinical specimens; 4 specimens
were taken from cerebrospinal fluid, 9 specimens from blood, 2 specimens from
umbilical swab. Laboratory strain, E. coli ATCC 25922, was included in
all susceptibility testing. Two K. pneumoniae strains were used in phenotypic
detection of ESβL; one of them harbours ESβL (positive control) and
the other strain did not produce ESβL (negative control).
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was determined by using E-test strips
(AB Biodisk, Solana, Sweden) on Mueller-Hinton agar (Oxoid, Basingstoke, England)
according to Clinical and Laboratory Standards Institute (Clinical Laboratory
Standards Institute, 2006) recommendations.
Phenotypic Detection of ESβL
ESβL was detected phenotypically using ceftazidime/ceftazidime-clavulanic
acid and cefotaxime/cefotaxime-clavulanic E-test strips. ESβL-producing
K. pneumoniae isolate showed a Minimum Inhibitory Concentrations (MICs)
by the antibiotics cefotaxime and ceftazidime in the presence of clavulanic
acid of more than 8 fold.
Preparation of DNA Template
DNA templates for Polymerase Chain Reaction (PCR) process, were generated
by suspending 5 colonies of overnight culture of K. pneumoniae isolate
grown on tryptone soy agar (Winlab, UK) in 200 μL of HPLC grade water (BDH,
England). The suspension was boiled at 100°C for 10 min in thermal block
(Techne, UK), then centrifuged at 15000 rpm for 5 min. An aliquot of 1 μL
of the supernatant was used as DNA template in the PCR.
PCR and DNA Sequencing Analysis
All 16 multiresistant K. pneumoniae isolates included in the study
were screened for alleles encoding the most frequently reported ESβLs in
Europe, Asia and USA, that is the blaCTX-M phylogenetic lineage
groups 1, 2, 8 and 9, the blaSHVs and the blaTEMs.
The PCRs for genes encoding TEM, SHV and CTX-M β-lactamases were performed
using primers and conditions described previously (Al-Agamy et al.,
2006; Bonnet et al., 2001; Nüesch-Inderbinen et al., 1997).
Four PCR experiments that differentiated between CTX-M-1, -2, -8 and -9 groups
of enzymes were performed for CTX-M-positive isolates using the primers and
the methods described by Baraniak et al. (2002), Chanawong et al.
(2002) and Villegas et al. (2004).
| Table 1: | The primers used in amplification of β-lactamases genes |
| F: Forward, B: Backward | |
The primers used in this work are shown in Table 1. The PCR products were purified using PCR purification kit (Qiagen, Hilden, Germany) and those products were sequenced on both strands using 3130XL genetic analyser, (Applied Biosystem) according to Sanger et al. (1977). PCRs used to detect bla genes were done in a thermocycler (Fexigene, Techne, UK). However, PCR machine used in sequencing was PCR system 9700, (Applied Biosystem).
Conjugation Experiments
Transferability was tested by Broth Matting Method using rifampicin-resistant
mutant E. coli JM105 (Pharmacia Biotech, USA) as a recipient at 1:2 donor/recipient
ratios (Vatopoulos et al., 1990). Conjugation mixtures were plated on
MacConkey agar containing 100 μg mL-1 rifampicin and 10 μg
mL-1 cefotaxime and incubated for 24-48 h at 37°C to select transconjugants.
The MIC and PCRs were done to the transconjugants.
RESULTS
Antimicrobial Susceptibility
The antimicrobial resistance pattern of K. pneumoniae is shown in
Table 2. The results indicate that all isolates of K. pneumoniae
were multidrug resistant. All isolates were resistant to amoxicillin, aztreonam,
cefotaxime, ceftazidime, cefotriaxon at MIC>256 μg mL-1,
ciprofloxacin (MIC>32 μg mL-1), amikacin (MIC128->256
μg mL-1) and gentamicin (MIC196->256 μg mL-1).
They were susceptible to cefoxitin (MIC 1-2 μg mL-1) and imipenem
(MIC 0.0625-0.125 μg mL-1). The susceptibility to cefepime varied
with MICs ranged from 16 to >256 μg mL-1, while 50% of the
isolates had MIC of>256 μg mL-1. The resistance to third
generation cephalosporins was markedly inhibited by clavulanic acid.
Characterization of ESβL
The results of phenotypic detection of ESβL revealed that all isolates
were found to be having ESβL.TEM, SHV and CTX-M β-lactamase genes
in ESBL producing K. pneumoniae isolates by PCR. CTX-M β-lactamase
gene was further investigated by PCR using the specific primers which amplify
CTX-M-1 group, CTX-M-2 group, CTX-M-8 group and CTX-M-9 group. The PCR results
showed that all isolates harbored CTX-M-1 like genes and no amplicons were obtained
for other three tested CTX-M groups.
| Table 2: | Susceptibility testvzing results of ESBL-producing Klebsiella pneumoniae isolates |
| Valuse in bracket are percentage | |
The purified PCR products of TEM, SHV and CTX-M-1 like genes were sequenced on both strands. The DNA sequencing of 16 isolates showed that they carried blaTEM-1, blaSHV-1 and blaCTX-M-15 genes.
Transferability of the Antibiotic Resistant Determinants
Matting out assay showed that transconjugants were obtained from the donor
strains when selected on MacConkey agar containing 100 μg mL-1
rifampicin and 10 μg mL-1 cefotaxime. The MIC of the transconjugants
revealed that they were resistant to cefotaxime and ceftazidime. However, the
transconjugants were sensitive to ciprofloxacin (MIC 0.06 μg mL-1).
The PCRs showed that all transconjugants carry TEM-1, SHV-1 and CTX-M-15 genes.
DISCUSSION
The dissemination of ESβL-producing Enterobacteriaceae in the hospital setting is a problem with major therapeutic and epidemiological consequences, particularly when the affected wards involve those caring for critically ill patients such as the ICU. Multiple outbreaks of ESβL-producing Enterobacteriaceae have been reported over the past 2 decades and K. pneumoniae has been shown to be the most frequently involved organisms (Rebuck et al., 2000; Quale et al., 2002). In the present study, we have characterized the strain causing the first hospital-associated outbreak of an ESβL-producing member of the Enterobacteriaceae family in Eastern province. Sixteen multiple resistant K. pneumoniae strains were isolated in 2007 when an outbreak occurred in NICU, Al- Qatif Hospital. Klebsiella pneumoniae isolates were found to be producing ESBL and multiple resistant to most of the tested antibiotics. However, they were susceptible to imipenem and cefoxitin. Imipenem is frequently the only therapeutic options available for treatment of hospital-acquired severe infections caused by multiresistant ESβL-producing K. pneumoniae such as the strain described in the present study. Nevertheless, universal susceptibility to these last-line antimicrobials in K. pneumoniae is no longer guaranteed. Indeed, several carbapenemases have been described as occurring in K. pneumoniae, including representatives from classes A, such as the highly disseminated KPC enzymes; B, such as IMP, VIM, GIM, SIM or SPM metallo-β-lactamases; or class D, such as OXA-48 (Yigit et al., 2001; Nordmann and Poirel, 2002; Poirel et al., 2004). Fortunately our strains were susceptible to imipenem and to cefoxitin and were devoid of carbapenemases and AmpC plasmid-mediated β-lactamases (Data not shown).
Genotypic characterization of β-lactamase genes revealed that ESBL producing K. pneumoniae isolates carry the blaTEM, blaSHV and blaCTX-M genes. Over the past decade, CTX-M genes have become the most common ESβLs in many countries all over Europe, Asia, Africa and America and most recently in Kuwait (Al-Agamy et al., 2006; Livermore et al., 2007; Ensor et al., 2009). All isolates carry CTX-M-1 like gene group whereas, no PCR amplified products were detected with other three CTX-M groups. The results of the PCR showed that these isolates harbored blaCTX-M-1-like gene, which includes blaCTX-M-1, blaCTX-M-3, blaCTX-M-10, blaCTX-M-12, blaCTX-M-15, blaCTX-M-22, blaCTX-M-23 and blaCTX-M-28 (Livermore and Woodford, 2007). DNA sequencing of TEM, SHV and CTX-M-1 like genes revealed that presence of blaTEM-1, blaSHV-1 and blaCTX-M-15. CTX-M-15, which was first detected in India in 2001 (Karim et al., 2001) and it is now recognized as the most widely distributed CTX-M enzyme (Livermore et al., 2007). The rapid and massive spread of CTX-M-type ESβLs is rapidly changing the ESβL epidemiology and, in some geographical areas, these enzymes are now the most prevalent ESβLs in Enterobacteriaceae. Outbreaks of CTX-M-15-producing Enterobacteriaceae have also been reported in France, Italy, Spain, Portugal, Austria, Norway, the United Kingdom, Tunisia, South Korea, Canada, Egypt and Kuwait (Conceição et al., 2005; Abbassi et al., 2006; Al-Agamy et al., 2006; Soge et al., 2006; Livermore et al., 2007; Ensor et al., 2009). In a recent study was conducted in Saudi Arabia, K. pneumoniae LO10 producing ESβL SHV-12 isolated during outbreak occurred in the neonatal unit at Security Forces hospital (Al-Obeid et al., 2008). However, in our study, K. pneumoniae producing ESβL CTX-M-15 isolated during outbreak occurred in NICU, Al-Qatif Hospital. The study performed by Al-Obeid et al. (2008) and the present study are the only two studies conducted that have describes the outbreaks in ICU due to ESβL-producing K. pneumoniae in Saudi Arabia. The variation in two studies is based on the type of ESβL while in the previous study it was the blaSHV-12 genes. However, in the current study the variation is in blaCTX-M-15 genes. This variation is difficult to explain, but may be due to differences in the geographical area, differences in the time of collection of isolates and differences in the type and volume of consumption of antibiotics. Outbreaks of Klebsiella infections in neonatal units associated with widespread colonization of babies have been reported (Hobson et al., 1996). Outbreaks of K. pneumoniae usually occurred by monoclonal however in fewer studies outbreak occurs by multiclonal (De-Oliveira et al., 2008; Carrër et al., 2009). In the current study, we can not confirm this outbreak due to single clone or multiple clones due to the lack of a genomic epidemiological marker. Techniques such as pulsed field gel electrophoresis and random amplified polymorph DNA has not been done to investigate this outbreak. However, there are great evidences to indicate that an outbreak of CTX-M-15 producing K. pneumoniae may be due to single clone. Some evidences were taken such as all isolates had the same resistance pattern, all isolates were able to transfer cefotaxime resistant determinants, antibiotic resistance of the transconjugants is similar and the isolates and their transconjugants had TEM-1, SHV-1 and CTX-M-15.
General considerations were taken by infection control practice in the NICU to stop the outbreak. Some of the important consideration; the isolation of all colonized and diseased patients as a cohort nursing and admission has stopped but unavoidable cases were sent to another unit; dedication of nurses to colonized and diseased patients; environmental and clinical samples was obtained. In addition, environmental specimens including bed, milk, milk bottles, equipment, thermometer, blood pressure cuff, intravenous saline, ventilators, respiratory equipment soap and water were taken. All environmental specimens were negative except blood pressure cuff was positive for K. pneumoniae which is sensitive to antibiotics. On the other hand, clinical specimens including umbilical, rectal, intravenous swab, finger print of nurses and those who were positive umbilical and rectal swab rescreened after decolonization with chlorohexadine and alcohol. Frequent meeting with pediatric head, NICU supervisor and house keeping department was on daily babies; decontamination of environment was routinely done; infection control course for all hospital nurses for a period of 2 months was mandatory.
In conclusion, this is the second study that describes the outbreak of ESβL-producing K. pneumoniae in Saudi Arabia and the first report of outbreak in Saudi Arabia due to blaCTX-M-15 genes. It is evident that the blaCTX-M genes are now increasingly detected in patients throughout Saudi Arabia and to define the extent of this spread, more studies on the cefotaxime-resistant Klebsiella sp. and other Enterobacteriaceae needs to be carried out in the different geographical area around the kingdom. Furthermore, outbreaks in Saudi hospitals must be investigated to define the predominate ESβL gene in the Enterobacteriaceae strains that are responsible for the outbreaks.
ACKNOWLEDGMENT
This study was supported by research grant number CPRC250, College of Pharmacy, Research Center, King Saud University, Riyadh, Saudi Arabia.