Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 130-135

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 130-135

Original Article

An outbreak of CTX-M-15-producing Klebsiella pneumoniae isolates in an intensive care unit of a teaching hospital in Kuwait

N Al Sweih¹, MF Salama², W Jamal¹, G Al Hashem², VO Rotimi¹

¹ Department of Microbiology, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 13110, Kuwait
² Infection Control Unit, Mubarak Al Kabir Hospital, P. O. Box 43787, Hawally 32052, Kuwait

Correspondence Address: N Al Sweih Department of Microbiology, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 13110 Kuwait nalsweih@hsc.edu.kw

Date of Submission: 11-Dec-2010
Date of Acceptance: 01-Feb-2011

Code Number: mb11031

PMID: 21654106

DOI: 10.4103/0255-0857.81791

Abstract

Keywords: CTX-M-15, ICU, Kuwait, Klebsiella pneumoniae, outbreak

Introduction

The increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-KP) isolates is of particular concern as these isolates often express resistance to multiple antibiotics and, as a result, complicate antibiotic therapy and interfere with empirical therapy. ^[1] Hospital outbreaks due to multidrug resistant K. pneumoniae isolates producing different ESBL types have been described throughout the world. ^[2],[3] A prevalence as high as 50% of nosocomial bloodstream infections caused by ESBL-KP has been reported in some countries in Europe and South America. ^[4] Derivatives of TEM and SHV enzymes constituted the majority of ESBLs in the 1980s and 1990s but have now been replaced by the CTX-M enzymes which have rapidly disseminated throughout the world. ^[5],[6],[7] CTX-M-15, the most expansive of these enzymes, was first found in isolates of Enterobacteriaceae from India but is now prevalent almost everywhere in the world. It appears to be associated with epidemic plasmids flanked with insertion sequences that facilitate easy spread and hyperproduction of β-lactamase which may, in part, explain the rapid spread of the enzyme.^[8]

In a previous report that studied the burden of CTX-M-15-producing Salmonella spp. in Kuwait and United Arab Emirates, Rotimi et al.^[9] found that 12.1% of their isolates were producers of this enzyme. Other recent experiences in Kuwait lent credence to the dramatic worldwide increase in the prevalence of CTX-M-15-type β-lactamase among the family of Enterobacteriaceae, in particular clinical isolates of Escherichia coli and K. pneumoniae. This assertion is supported by finding CTX-M-15 as the most common CTX-M-type enzyme in E. coli in Kuwait, ^[10] a finding which confirmed the earlier report by Ensor et al. ^[7] In our experience, CTX-M-15-producing K. pneumoniae causing outbreaks of infections is rare in Kuwait and, rarer still, are those associated with high mortality.

In the present study, we investigated a nosocomial outbreak of multi-resistant ESBL-producing isolates of K. pneumoniae in the adult intensive care unit (ICU) of our hospital.

Materials and Method

Setting

Our hospital is a 531-bed tertiary university-affiliated hospital with a catchment population of over 1 million people. It has a compliment of general medical, urology, paediatrics, surgical, and dialysis wards, paediatric and adult ICUs, and outpatient departments. The adult ICU comprises 2 wings of 12 beds each in addition to 3 separate isolation rooms.

Definition

K. pneumoniae-positive cases were defined as those patients admitted to the ICUs from whom at least one clinical sample recovered during the ICU stay contained an ESBL producer together with clinical evidence of infection. Episodes of clinical infection were considered acquired in the ICU if they appeared 48 h after ICU admission. Standard Centers for Disease Control and Prevention (CDC) criteria were used to carefully define healthcare-associated infections. ^[11]

Bacterial identification and antibiotic susceptibility testing

All the isolates were identified as K. pneumoniae by the API 20E system (bioMérieux Marcy-řÉtoile, France) and VITEK 2 ID System (bioMérieux). Antibiotic susceptibility testing against amikacin, amoxicillin-clavulanic acid, ampicillin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, imipenem, meropenem, piperacillin, piperacillin-tazobactam, and tigecycline was performed using the E-test (AB Biodisk, Solna, Sweden) method according to the manufacturer′s protocol. E. coli ATCC 25922 was included in each run for quality control. Results were interpreted according to the recommendations of the Clinical Laboratory Standard Institute (CLSI) ^[12] and US FDA tigecycline breakpoint of ≤2 μg/ml [Tygacil package insert (June 2005), Wyeth Pharmaceuticals Inc., Philadelphia, PA, USA].

Detection of extended-spectrum ß-lactamases and DNA extraction

The ESBL strips (AB Biodisk) consisting of cefotaxime (CT)/cefotaxime combined with clavulanic acid (CTL) and ceftazidime (TZ)/ceftazidime combined with clavulanic acid (TZL; AB Biodisk) were used to identify the ESBL-producing strains according to the manufacturer′s protocol. Quality assurance was performed for the isolates using ATCC strains: E. coli ATCC 25922 (ESBL-negative), K. pneumoniae ATCC 700603 (ESBL-positive), and Pseudomonas aeruginosa ATCC 27853 control for ceftazidime.

All the isolates were cultured on blood agar and incubated at 37°C for 24 h. A QIAmp DNA Minikit (QIAGEN GmbH, Hidden, Germany) was used for the extraction of DNA according to manufacturer′s manual protocol C for the isolation of genomic DNA from bacterial culture.

Identification of β-lactamase genes and insertion sequences

Genes encoding the ESBLs and other β-lactamases were detected by PCR using published specific primer pairs for bla_CTX-M , bla_SHV , and bla_TEM genes as follows: universal consensus primers, MA-1 5′-SCS ATG TGC AGY ACC AGT AA-3′ and MA-2 5′-CCG CRA TAT GRT TGG TGG TG-3′ (for bla_CTX-M ), ^[13] OS-5 5′-TTA TCT CCC TGT TAG CCA CC-3 and OS-6 5′-GAT TTG CTG ATT TCG CTC GG-3′ (for bla_SHV ), and C 5′- TCG GGG AAA TGT GCG CG-3 and D 5′-TGC TTA ATC AGT GAG GCA CC-3′ (bla_TEM ). ^[14] OXA-1 and insertion sequence ISEcp1 were detected using primers specific for bla_OXA-1 A, 5′-GGA TAA AAC CCC CAA AGG AA-3; bla_OXA-1 B, 5′-TGC ACC AGT TTT CCC ATA CA-3′; ^[15] and insertion sequence ISEcp1A, 5′-GCAGGTCTTTTTCTGCTCC-3′; ISEcp1B, 5′-ATTTCCGCAGCACCGTTTGC-3′. ^[16] Cycling conditions were as follows: Initial denaturation at 94°C for 3 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s followed by a final elongation at 72°C for 5 min. ^[17] Both strands of the PCR products were sequenced with an automated sequencer (ABI 300; Applied Biosystems, Foster, CA, USA). Analysis of sequences was carried out with software available at the internet web page http://www.ncbi.nlm.nih.gov .

Transfer of resistance phenotypes

Transfer experiments were carried out between the CTX-M-producing K. pneumoniae donors and recipient E. coli J53 rif ^r as previously described. ^[18] The conjugative transfer of CTX-M to E. coli J53 was performed on nutrient agar (NA) plates with culture grown into the logarithmic phase. Transconjugants were selected on NA supplemented with 100 μg/ml rifampicin (Sigma-Aldrich) and 2 μg/ml cefotaxime.

Genomic fingerprinting by PFGE

The isolates were fingerprinted by pulsed-field gel electrophoresis (PFGE) typing of the extracted whole-cell genomic DNA embedded in 1% agarose plugs and digested with Xba1. The Xba1-digested genomic DNA was electrophoresed in a 1% certified agarose gel with a voltage gradient of 6 V/h at 14°C at an angle of 120° using the CHEF-MAPPER XI System (Bio-Rad Laboratories, Hercules, CA, USA). Banding patterns were analyzed using FPQuest ^TM software (Bio-Rad Laboratories) and strains defined as having PFGE profiles of >94% similarity. A lambda ladder (New England Biolabs, Beverly, MA, USA) was included in each gel run. DNA relatedness was estimated using the criteria of Tenover et al .^[19]

Result

Outbreak and intervention

An outbreak of ESBL-KP in 14 patients within 2 months was encountered in the medical ICU of our teaching hospital. Nine patients had bloodstream infections (BSI; isolates 1-3, 5-9 and 14), three urinary tract infections (UTI; 10, 11 and 13) and two pulmonary infections (PI; 4 and 12). The profiles of the patients including source of the isolate, date of isolation, underlying disease and treatment are shown in [Table - 1]. The ages of the patients ranged from 24 to 89 years (mean 59.5 ± 12.6 years). The time of admission to isolation of the ESBL-KP ranged from 3 to 43 days with an average of 15.8 days. A mortality rate of 21.4% was recorded in patients whose cause of death was overwhelming septicaemia and septic shock.

The index case was a 53-year-old male patient admitted on 28 June 2008 as a result of myocardial infarction (MI) complicating cardiovascular accident (CVA). He presented on 3 August with fever, septicaemia and subsequent septic shock. His blood culture yielded ESBL-KP susceptible only to carbapenems and tigecycline. He was treated with meropenem and tigecycline but died 12 days later. The second case was a patient with obstructive jaundice and pancreatitis admitted on 29 June and initially nursed on a bed next to the index case. He presented with high fever 8 days after the isolation of ESBL-KP from the index case. His blood culture taken on 10 August yielded a phenotypically similar organism. He was promptly moved to an isolation room and treated with meropenem but died 2 days later. Within the next 3 weeks, phenotypically identical ESBL-KP were isolated from six patients, three of whom were positive on the same day, 17 August. They presented with septicaemia (five patients) and pneumonia complicated by pancytopaenia and anaemia (one patient). Despite reinforcing infection control measures, the epidemic strain reappeared 3 weeks later with a spate of infections in another set of 6 patients within 6 days of each other during 20-25 September [Table - 1]. The total number of K. pneumoniae strains isolated in the ICU in 2008, including the 14 outbreak strains, was 19; the 5 non-outbreak isolates were susceptible strains.

Patients, staff members and ICU environment were screened. Control measures instituted included isolation of some patients in isolation rooms as well cohort nursing the others in a localized area in the ICU under the supervision of dedicated nurse. Multi-component intervention included extensive decontamination of the ICU environment three times daily, with initial cleaning with warm water followed by disinfection with a hypochlorite solution (1000 ppm), continued ICU personnel educational programmes, rigorous open surveillance of adequate compliance with barrier precautions, the use of disposable gloves and aprons, and careful disinfection of hands with soap and water and hydro-alcohol solution before and after patient care. Cleaning protocols and housekeeping procedures were revised. Compliance with the antibiotic policy was monitored by the clinical microbiologists. Adequate compliance with the control programme was supervised by two specially designated members of the infection control team (one physician and one nurse) who attended the ICU daily after the implementation of the interventional programme in August 2008.

Results of patients, personnel (fingers, rectal and throat swabs) and environmental screening were negative but a swab specimen obtained from suction equipment in a room of one of the infected patients yielded an identical ESBL-KP.

Antibiotic susceptibility and resistance transfer

The 14 clinical isolates were resistant to all the β-lactam antibiotics (MICs, >256 μg/ml) tested, except for carbapenems (MICs, 0.002-1 μg/ml), ciprofloxacin (MIC, >32 μg/ml) and amoxicillin-clavulanic acid (MICs, 6-12 μg/ml). Tigecycline (MICs, 0.032-2 μg/ml) had excellent activities against all isolates. Strains isolated from the suction pump (isolate 15) had the same resistance pattern as the others.

Transconjugants created in E. coli J53 from all the donors were shown by PCR to contain bla_CTX-M-15 and bla_TEM-1 . MICs of cefotaxime and ciprofloxacin for the transconjugants and recipient strains were >256 and >32 μg/ml and 0.064 and 0.012 μg/ml, respectively.

Sequencing analysis and clonality of isolates

All isolates were positive for CTX-M β-lactamase [Figure - 1]. The DNA sequencing revealed identical sequences belonging to CTX-M-15. They also expressed blaTEM ; sequencing on positive PCR products of the same isolates showed that they carried the bla_TEM-1 gene. None of the 15 isolates was positive for bla_SHV , bla_OXA-1 , or bla _ISEcp1 frequently identified upstream of the bla_CTX-M genes.{Figure 1}

The digestion of genomic DNA with Xba1 produced 10-13 fragments. The 15 isolates gave a related PFGE pattern. DNA restriction analysis shown in the dendogram [Figure - 2] distinguished two patterns (clones A and B) and corresponding bands of the same apparent size all showing close relatedness with a Dice coefficient of >94.1% similarity.

Discussion

Our report highlights the spread of clonally related CTX-M-15-producing K. pneumoniae in the adult ICU of a teaching hospital in Kuwait. Both the clinical and microbiological data show that the infections were acquired in the ICU. There was an overlapping ICU stay which was probably involved in the spread of the epidemic strain from one patient to the other as a result of contamination of the hands of healthcare providers, or medical equipment, corroborated by the isolation of ESBL-producing K. pneumoniae from the suction pump located in the room of an infected patient.

The DNA amplification of the ESBL genes revealed that the K. pneumoniae isolates produced only CTX-M-15 and non-ESBL β-lactamase (TEM-1). CTX-M-15 β-lactamase, first detected in India in 2001, ^[18] is one of the most frequently reported ESBLs produced by various species of Enterobacteriaceae worldwide, including Kuwait. ^[5],[7],[20] Several reports have confirmed the emergence of scattered CTX-M-15-producing K. pneumoniae isolates causing outbreaks in adult and neonatal ICUs in Sweden, ^[20] Saudi Arabia, ^[21] France, ^[22] Madagascar, ^[23] and Croatia. ^[24]

Experience with this epidemic strain appears to have been limited to the 2-month period in 2008. However, our concern regarding the possibility of future outbreaks is predicated on the fact that K. pneumoniae is a very efficient hospital pathogen and, in addition, can readily acquire plasmid-mediated CTX-M-15 genes from E. coli.

A PFGE study is an excellent method of determining clonality of ESBL-producing K. pneumoniae isolates and it was used to confirm that the outbreak was caused by two closely related clones. We can speculate at this time that these isolates are essentially the same clone which had mutated to produce a related subclone. Outbreaks of MDR K. pneumoniae are usually monoclonal although a few studies have reported multiclonalilty of epidemic strains causing outbreaks. ^[21] It is conceivable that the epidemic strain had undergone spontaneous mutation in the gut of some of these long-stay patients resulting in polyclonality and antibiotic resistance in the critically ill. ^[25] Although no K. pneumoniae was isolated from the stool or throat of these patients, a prolonged stay in the ICU particularly of the index case and some others and the severity of the underlying illnesses might have played a role in the evolution of the second clone.

Dedicated and robust infection control measures used during the period no doubt helped in stemming the outbreak resulting in complete eradication of the MDR strain.

In conclusion, we have documented an outbreak caused by CTX-M-15-producing K. pneumoniae in an adult ICU which is the first detailed report of such outbreak in an adult ICU in Kuwait. Our report also showed that stringent infection control practices when meticulously followed can stop and prevent a repeat of similar outbreaks.

References

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21.	Al-Agamy MH, Shibl AM, Tawfik AF. Prevalence and molecular characterization of extended- spectrum beta-lactamase-producing Klebsiella pneumoniae in Riyadh, Saudi Arabia. Ann Saudi Med 2009;29:253-7.  Back to cited text no. 21  [PUBMED]
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23.	Randrianirina F, Vedy S, Rakotovao D, Ramarokoto CE, rasitohaina H, Carod JF, et al. Role of contaminated aspiration tubes in nosocomial outbreak of Klebsiella pneumoniae producing SHV-2 and CTX-M-15 extended-spectrum â-lactamases. J Hosp Infect 2009;72:23-9.  Back to cited text no. 23
24.	Vranic-Ladavac M, Bosnjak Z, Beader N, Barisic N, Kalenic S, Bedenic B. Clonal spread of CTX-M-15 producing Klebsiella pneumoniae in Croatian hospital. J Med Microbiol 2010;59:1069-78.  Back to cited text no. 24
25.	van Saene HK, Taylor N, Damjanovic V, Sarginson RE. Microbial overgrowth guarantees increased spontaneous mutation leading to polyclonality and antibiotic resistance in the critically ill. Curr Drug Targets 2008;9:419-21.  Back to cited text no. 25

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