Spread of colistin resistant non-mucoid Pseudomonas aeruginosa among chronically infected Danish cystic fibrosis patients

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1 Journal of Cystic Fibrosis 7 (2008) Spread of colistin resistant non-mucoid Pseudomonas aeruginosa among chronically infected Danish cystic fibrosis patients Helle Krogh Johansen a,b,, Samuel M. Moskowitz c, Oana Ciofu b, Tacjana Pressler a, Niels Høiby a,b a Department of Clinical Microbiology, Dept and Danish Cystic fibrosis Centre, Dept. 5003, Rigshospitalet, Copenhagen, Denmark b Institute of International Health, Immunology and Microbiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark c Division of Pulmonary Medicine, Children's Hospital and Regional Medical Centre and University of Washington School of Medicine, Seattle, Washington 98195, USA Received 30 September 2007; received in revised form 27 January 2008; accepted 4 February 2008 Available online 20 March 2008 Abstract Background: Colistin resistant Pseudomonas aeruginosa have rarely been reported in cystic fibrosis (CF) patients. Methods: We performed a 17-year prospective study on colistin susceptibility and compared our findings with clinical variables. Results: The first outbreak started in 1995 and lasted 5 years. It involved 27 CF patients who had inhaled colistin twice daily for a median of 10 years. Colistin resistant isolates persisted in individual patients for a median of 75 days after colistin was withdrawn. A second outbreak started in It involved 40 patients, 17 of whom were the same as in the first outbreak. Most resistant isolates belonged to two major clones that had similar genotypes in the two outbreaks. The P. aeruginosa isolates were all non-mucoid and they appeared in a group of chronically infected patients that had been admitted to the same ward for antibiotic treatment and had been followed at the same week-days in the outpatient clinic. Patients were individually isolated to avoid cross-infection and colistin inhalation was avoided in the CF outpatient clinic and in the ward after both outbreaks. Since 2004, no further spread has been observed. Conclusion: It is important that the colistin resistant clones do not spread to non-infected patients since colistin is an important antibiotic for eradication of initial and intermittent P. aeruginosa colonisation European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Colistin resistance; P. aeruginosa; Cystic fibrosis; Outbreak 1. Introduction Chronic Pseudomonas aeruginosa lung infection develops in most patients with cystic fibrosis (CF) and is usually preceded by Preliminary data were presented at the 26th European Cystic Fibrosis Conference, Belfast, Ireland, June 4 7, 2003 and at the 17th Annual North American CF Conference, Anaheim, California, U.S.A., October 16 19, 2003 and were published in abstract form in Pediatr. Pulmonology 2003; Suppl. 25, 304. Corresponding author. Department of Clinical Microbiology 9301, Rigshospitalet, Juliane Maries, Vej 22, DK-2100 Copenhagen Ø, Denmark. Tel.: x6411, fax: address: hkj@cochrane.dk (H.K. Johansen). a stage of intermittent colonisation [1]. Once the bacteria have established a chronic biofilm infection in the lungs, they can hardly ever be eradicated, although aggressive therapy with different antibiotic combinations and different routes of delivery can temporarily postpone the progression of the lung disease and prolong the life of the patients [2,3]. Since 1984, all chronically infected Danish CF patients have inhaled 2 million units of polymyxin E (colistin, 1 mg colistin base = 30,000 units) twice daily between the regular intravenous antibiotic courses in order to maintain pulmonary function and decrease chronic inflammation within the respiratory tract [4]. Moreover, in an attempt to postpone chronic infection, the routine use of early aggressive treatment (inhaled colistin and oral ciprofloxacin for three weeks /$ - see front matter 2008 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi: /j.jcf

2 392 H.K. Johansen et al. / Journal of Cystic Fibrosis 7 (2008) to three months) for CF patients intermittent colonised with P. aeruginosa was introduced in 1989 [5,6]. Polymyxins are cyclic cationic antimicrobial peptides that are highly active against many Gram-negative bacteria, including P. aeruginosa. They exert their bactericidal activity through disruption of the cell membrane leading to leakage of cell contents and cell death. This is achieved at least in part by binding to lipopolysaccharide (LPS), a major component of the Gramnegative cell surface, through interactions with phosphates and fatty acids of LPS core and lipid A moieties [7,8]. The operon pmrh-m is directly controlled by pmrab, and the gene products are responsible for the synthesis of N 4 -aminoarabinose that binds to lipid A and reduces binding of polymyxins to LPS, whereby resistance arises [9]. Although outbreaks of multi-resistant P. aeruginosa have been reported in other settings [10], there has, to our knowledge, been only one paper, from the United Kingdom, reporting a small outbreak of colistin resistant P. aeruginosa among patients with CF [11]. This is surprising because colistin is commonly used for inhalation and intravenous therapy in CF patients worldwide [12 15]. This paper reports on a biphasic spread of colistin resistant non-mucoid P. aeruginosa among chronically infected CF patients in our centre. The course of the outbreak was presumably facilitated by the daily use of colistin inhalation for the intensive treatment of chronic P. aeruginosa infections [16]. 2. Patients and methods 2.1. General care The Copenhagen CF Centre was established in 1968 [17,18]. Since 1971 all patients have been followed monthly in the outpatient clinic for clinical status, pulmonary function and bacteriological investigations of lower respiratory tract secretions. From 1976, all patients with chronic P. aeruginosa infection have been treated electively every 3 months with a 2-week intravenous antibiotic course. Cohort isolation was introduced in 1981 and further expanded in 1982 subsequent to an epidemic spread of a multi-resistant non-mucoid strain of P. aeruginosa among patients with chronic P. aeruginosa infection [19], who were hospitalized in the same ward for regular i.v. courses of anti-pseudomonal chemotherapy [20]. The cohorts consist of 1) patients chronically infected with multi-resistant P. aeruginosa (i.e. resistant to two or more different antimicrobial classes) [19], 2) patients chronically infected with P. aeruginosa with natural, wild-type susceptibility to antibiotics, 3) patients without chronic P. aeruginosa infection including patients with intermittent P. aeruginosa colonisation [2] and 4) patients harbouring either Achromobacter xylosoxidans or the Burkholderia cepacia complex are individually isolated from all other CF patients. Patients are segregated in the outpatient clinic, in the wards, and at social events, such as summer and winter camps. This policy has successfully reduced the incidence of P. aeruginosa colonization and infection [21] and maintained a low incidence of bacteria belonging to A. xylosoxidans and the B. cepacia complex [22] Colistin policy To promote the goal of maintaining lung function and decrease inflammation, treatment of all patients with chronic P. aeruginosa lung infection with inhaled colistin (2 million units twice daily) between regular intravenous antibiotic courses was adopted as standard practice at the Danish CF centre in Copenhagen in 1984 [4]. Since 1989, all intermittent colonised patients have been treated with oral ciprofloxacin in combination with colistin inhalation, in the beginning for 3 weeks, and since 1995 for 3 months (early eradication) every time P. aeruginosa was isolated from the respiratory tract, to prevent or delay onset of chronic infection [23]. Colistin was also from time to time used intravenously while the chronically infected patients were admitted to the hospital for treatment. In 1995, after 11 years of colistin use, colistin resistant non-mucoid P. aeruginosa were identified for the first time in the chronically infected population. In patients from whom resistant strains had been isolated, we adopted a policy of discontinuing colistin inhalation and replacing this with tobramycin inhalation until the resistant strain had not been cultured from the patients sputum for 6 months. This strategy has successfully been used to combat a previous epidemic of multi-resistant non-mucoid P. aeruginosa in our clinic [19]. Furthermore, patients harboring colistin resistant strains were isolated from the other CF patients for 6 months. Isolation arrangements were ceased after the patient had been free from colistin resistant P. aeruginosa for a period of 6 months. To decrease selection pressure, the use of inhaled colistin was prohibited in the CF ward [19] Microbiology Since 1968, when the CF centre in Copenhagen was established, sputum samples have been investigated every month by Gram-stained smears and aerobic cultures on selective media. These media include a Sabouraud plate, a 7% NaCl plate, a B. cepacia plate containing colistin and gentamicin and a blue plate (modified Conradi Drigalski's medium) selective for Gram-negative rods and non-selective media including 5% Danish blood agar and chocolate agar. Isolated bacteria were identified as described previously [2]. Direct plating of sputum samples on a 14 cm blood agar plate (State Serum Institute, Copenhagen, Denmark) with discs containing anti-pseudomonas antibiotics including colistin are used for primary susceptibility testing and for detection of resistant mutants [2]. In 1991, we decided to collect non-mucoid and mucoid P. aeruginosa isolates from all the CF patients in our clinic every second year to follow the development of resistance to all antibiotics used in the CF ward. The antibiotics include colistin, tobramycin, meropenem, ciprofloxacin, aztreonam, piperacillin/tazobactam and ceftazidime. The minimal inhibitory concentration (MIC) determination on the collected isolates is done by the agar dilution method on Müller Hinton agar. This method differs from the monthly routine susceptibility testing of the sputum samples in the laboratory, where the agar diffusion method is used on Danish blood agar plates (Statens Serum Institut, Copenhagen, Denmark), employing Neosensitabs (Rosco, Roskilde, Denmark) [24]. A

3 H.K. Johansen et al. / Journal of Cystic Fibrosis 7 (2008) colistin resistant isolate is suspected in the routine laboratory when the zone diameter around a polymyxin-containing disc (150 μg polymyxin) is b 18 mm (MIC N 32 μg/ml) Patient data The epidemiological, microbiological and clinical data were collected retrospectively from the patients' medical records. The data used in the present study include: age of the patient, gender, CF genotype, age at onset of chronic P. aeruginosa lung infection, number of years in treatment with colistin, doses, alternative treatments, number of years with chronic P. aeruginosa lung infection before acquisition of colistin resistant P. aeruginosa, age at acquisition of colistin resistant P. aeruginosa, number of days to clearance of resistant isolates, lung function and precipitating antibodies. In 7 selected patients who had colistin resistant P. aeruginosa in 1995 and from whom we also cultured the resistant isolate in 1997 or 1999 and again in 2004, the development in lung functions variables were evaluated over time, to detect possible adverse effects on lung function after long-term infection with colistin resistant P. aeruginosa Pulsed-field-gel-electrophoresis (PFGE) PFGE was used for bacterial genotyping and for identifying clonality of colistin resistant P. aeruginosa isolates. We used Spe I as restriction enzyme according to the methods described previously [25,26] (Fig. 2). PFGE patterns were compared visually and on the Gel Doc XR (Bio-Rad) and evaluated using the criteria previously published [25 27]. Isolates with differences in zero, one or two bands were considered identical; other isolates were considered unrelated Mechanism of resistance Mass spectral analysis of the lipid A moiety of lipopolysaccharide revealed that all strains lacked 2-hydroxylation of laurate but had aminoarabinose added [8] Statistics A one-sample t-test was used for analyzing within-patient changes. 3. Results All colistin resistant P. aeruginosa isolates in the first and second outbreak were of the non-mucoid phenotype. The patients who had colistin resistant isolates in the first outbreak had been chronically infected with P. aeruginosa for a median of 16 years (range 4 24 years). They had been treated with inhaled colistin for a median of 10 years (range 5 15 years), and one had, in addition, been treated with intravenous colistin for 16 days. The first colistin resistant P. aeruginosa isolate emerged in our clinic in A total of 27 patients had colistin resistant P. aeruginosa in the first outbreak ( ), 19 patients had a colistin resistant P. aeruginosa in a single year only, either in 1995, 1997 or 1999, whereas eight had growth in multiple years (Table 1). Only one of the patients had a colistin resistant isolate in all three years. The median age of the patients was 24 years (range 9 37 years) and they were infected for a median of 75 days (7 790 days). Based on PFGE two large clusters and seven individual strains were identified. The large clusters comprised 16 (cluster 1) and 5 patients (cluster 2), respectively (repetitions not included) (Fig. 1). In the first outbreak, no significant change in lung function as defined by FEV 1 or FVC% predicted (p=0.98 and p=0.43, respectively) or in P. aeruginosa precipitating antibodies (p=0.44) was observed, when compared to the patients' own lung function and precipitins one year before colistin resistance was discovered till one year after the patients had cleared their resistant isolates. Twenty-one of the 27 patients (78%) were ΔF508 homozygotes, 4 were ΔF508 heterozygotes and 2 patients had an unknown genotype. This distribution fits with the overall genetic composition in our clinic. In the period 2000 to 2004 we did not detect any colistin resistant P. aeruginosa neither in our routine laboratory nor by the biannually MIC investigations. However, during 2004 we realized that this particular strain had reemerged in our clinic. We found colistin resistant P. aeruginosa in 23 patients who had never been infected with the resistant isolate and in 17 patients from whom we had growth of colistin resistant P. aeruginosa in the first outbreak period. This corresponds to 14% of all our CF patients (40 of a total of 280) and 42% of all patients chronically infected with P. aeruginosa (40 out of 95 patients). In contrast to the first cohort, these newly colonised patients had been chronically infected with P. aeruginosa for a much longer time period (median 26 years, range 9 33 years) and they were a median of 8 years older at the time of infection (median Table 1 Number of patients infected with colistin resistant non-mucoid P. aeruginosa in each year, and number of patients infected with the two major cluster types and with individual clones Year Number of patients infected with colistin resistant non-mucoid P. aeruginosa Number of patients with chronic P. aeruginosa lung infection Number of patients in the CF centre , 2, (4 colonised in 1995) , 3, (1 also colonised in 1995, 3 also colonised , 3, 3 in 1997, 1 also colonised in 1995 and 1997) newly colonised and 17 previously colonised , 13, 6 a a 6 isolates were not genotyped due to technical difficulties, e.g. bacterial death. Number of patients infected with cluster 1, 2 or with individual clones

4 394 H.K. Johansen et al. / Journal of Cystic Fibrosis 7 (2008) At the most recent MIC investigations of all P. aeruginosa isolates from chronically infected CF patients (2006 and 2007), none of the patients had cleared the colistin resistant Pseudomonas and no new patients had become colonised. 4. Discussion Fig. 1. Pulsed-field-gel-electrophoresis (PFGE) pattern of 12 colistin resistant non-mucoid Pseudomonas aeruginosa isolates from cluster 1 in the first outbreak ( ), from 10 CF patients. The Spe restriction enzyme was used. Lane 1: molecular-weight standard, lane 2: pt #1, lane 3+4: pt #2 lane 5: pt #3, lane 6: pt #4, lane 7+8: pt #5, lane 9: pt #6, lane 10: pt #7, lane 11: pt #8, lane 12: pt #9 and lane 13: pt #10, K (control): PAO1. 32 years, range years). They had been treated with inhaled colistin for a median of 17 years (range years). The CF genotype distribution was similar to the overall genetic distribution in our clinic, 18 of the 23 patients were ΔF508 homozygotes (78%) and 5 were ΔF508 heterozygotes. There was a slight median decline in P. aeruginosa precipitating antibodies of one (range 0 to 5) among patients infected in 2004, from one year before to one year after colistin resistant P. aeruginosa was identified (p b 0.001). The majority of the isolates in the second outbreak belonged to the two large clusters (cluster 1: 6 patients and cluster 2: 8 patients) detected in the initial outbreak and three individual strains. Patients continued to be infected with isolates that belonged to the same cluster over time, and no changes from one cluster to another were seen. In contrast to the first outbreak, patients in the second outbreak had a significant decline in lung function as defined by FEV 1 or FVC% predicted (p b0.001 and pb0.003, respectively) when compared to the patients' own lung function one year before colistin resistance was discovered. In order to show possible adverse effects of being colonised for a longer period of time with the colistin resistant P. aeruginosa we also looked at FEV 1 or FVC% over time in a small group of 7 patients who were infected with colistin resistant P. aeruginosa in the first outbreak in 1995 and also infected in 2004 when the same isolate reoccurred. We found that they had a median decline in FEV 1 of 1.1% (range +0.8 to 2.7%) per year during the 10 years where they had been intermittently infected with the colistin resistant isolate and a median decline in FVC of 1.3% (range +0.2 to 3.5%) per year. The MICs for the colistin resistant isolates varied between 50 μg/ml and 400 μg/ml in the first outbreak and between 100 μg/ml and 400 μg/ml in the second outbreak. The geometric mean MIC of colistin of the non-mucoid susceptible isolates varied from 4.2 to 6.1 μg/ml and for mucoid isolates from 1.6 to 2.5 μg/ml from 1995 to 2005, respectively. The overall susceptibility pattern of the colistin resistant isolates against other anti-pseudomonal antibiotics used routinely in the CF ward did not differ when compared to the colistin susceptible non-mucoid isolates. Outbreaks of multi-resistant Gram-negative bacteria as well as transmission between CF patients have been reported before [11,22,28]. The present study underlines that although the patients are cohort isolated according to the susceptibility of the infecting P. aeruginosa there is a risk of other emerging infections within the cohorts [29,30], since it is known that exchange of strains with selective advantages occur within the CF cohort [19,31,32]. This biphasic outbreak has been going on for more than 12 years and in spite of cohort isolation of the patients since 1981 new outbreaks can still occur. There was long follow-up after this outbreak and fortunately, the clinical consequences for the patients were small. However, transmission of resistance genes to mucoid P. aeruginosa may occur and could have more serious consequences for the patients, as well as spread to newly colonised patients who are treated with inhaled colistin for their initial and subsequent eradication therapy. Resistance to colistin can occur either by inducible physiological resistance (tolerance) in response to membrane stresses such as divalent cation limitation or polymyxin exposure (colistin) or constitutively due to mutations in pmrb [8,33]. In both cases the mechanism is mediated through addition of an aminoarabinose to lipid A in the P. aeruginosa LPS molecule. The physiological resistance requires presence of colistin and is based upon a pmrab two component regulatory system, whereas the constitutive expression leads to stable production of aminoarabinose LPS not depending upon the presence of colistin [8]. When the colistin resistant P. aeruginosa isolates first appeared in our routine laboratory, we did not pay much attention to it. It had been suggested that colistin resistance is reversible and develops only rarely [34] and that resistance to polymyxin acquired in vitro probably has no relevance to the clinical situation, in which strains of P. aeruginosa have retained their susceptibility in hospital environments where the antibiotic has been much used [35]. We perform monthly routine susceptibility testing of the sputum samples in the laboratory by the agar diffusion method on Danish blood agar plates. The blood agar medium contains 0.72 mmol/l of calcium chloride and 0.59 mmol/l of magnesium sulfate [36]. In contrast, we measure MICs biannually on Müller Hinton agar that has a content of 2.05 mmol/l calcium chloride and 2.15 mmol/l magnesium sulfate. It has been shown that high degrees of resistance to polymyxin can be obtained by growing P. aeruginosa in a magnesium-deficient medium or in a calcium rich medium [34]. The difference in calcium and magnesium contents between the two culture mediums may explain the different susceptibility findings. When we culture the isolates on blood agar plates they are susceptible to colistin whereas using Müller Hinton agar the MIC of the isolates are 2 3 fold higher to colistin. A similar problem concerning P. aeruginosa and susceptibility to aminoglycosides has been published

5 H.K. Johansen et al. / Journal of Cystic Fibrosis 7 (2008) previously. It was found that resistance to gentamicin increased directly with the concentration of ionized magnesium in the medium and that both ionized calcium and soluble magnesium fractions control the inhibition zone sizes [37]. Fortunately, all the colistin resistant P. aeruginosa we have detected till now are non-mucoid. One reason for only detecting non-mucoid resistant strains may be that they are mainly located in the conductive zone of the respiratory tract where most of the inhaled colistin is deposited [38] and therefore the selective pressure on non-mucoid strains is higher than on mucoid strains [39,40]. Furthermore, in mucoid isolates the uronic acid of alginate binds cationic antibiotics [41]. In a study from our centre it was shown that 73 CF patients who were followed from three years before chronic P. aeruginosa infection to 10 years after chronic infection with non-mucoid strains had progression of the lung disease that was similar to patients not infected with P. aeruginosa [19,42]. This observation may explain why we did not see any decrease in lung function in the first cohort of colonised patients. In the second cohort where the patients did not clear the resistant isolates we found a significant decline in lung function after colistin resistant P. aeruginosa was contracted. This may in part be explained by the age of this cohort, as the second cohort was as a median eight years older that the first cohort and had been chronically infected with P. aeruginosa for a median of 10 years longer than the first cohort, and the avoidance of colistin inhalation may also have caused a more pronounced CF associated lung disease. However, the loss of lung function in the two groups did not exceed the yearly median slope of decline of 4% for FEV 1 and 3% for FVC for non-azithromycin treated patients at that time [43]. The group of patients followed over 10 years who harboured colistin resistant P. aeruginosa isolates for several years only had a minor loss of lung function over time. One reason for the successful adaptation of the colistin resistant P. aeruginosa among our CF patients could be that during a two-week course of anti-pseudomonal β-lactam antibiotic therapy, the sensitive bacterial population decreases in contrast to the pre-existing resistant sub-population [28]. It seems most likely that the colistin resistant isolate was selected during treatment and originated from a pre-existing resistant sub-population in the lungs of the patients. All patients in the outbreaks had been treated every day with inhaled colistin for at least 5 years, and small bacterial subpopulations may emerge during daily inhalations and exchange of resistant isolates may happen when patients are seen in the outpatients' clinic or admitted to the hospital ward for 14 days of intravenous antibiotic chemotherapy (Fig. 2). In 1995, when we first identified colistin resistant P. aeruginosa, it was limited to relatively few CF patients, but when it reemerged in 2004 it had spread to approximately 40% of all patients chronically infected with P. aeruginosa. A similar development has been reported from our centre in the mideighties where there was an epidemic of a non-mucoid P. aeruginosa resistant to aminoglycosides, carbenicillin, ureidopenicillins, ceftazidim, cefsoludin and imipenem. The isolate was present in the centre at a low frequency in 1973 but at that time it was susceptible to most antibiotics. However, during therapy with ceftazidime the MIC of ceftazidime increase, and high level of resistance to carbenicillin developed [19]. In both cases, emergence of resistance suddenly appeared after intensive selective pressure by antibiotics and it is most likely that colistin resistance was induced by the intensive use of this drug at the hospital and in the patients' home. Two identical clusters have dominated both outbreaks ( and 2004-) and when a patient was infected with one particular cluster, it remained the same when the patient had a colistin resistant isolate at a subsequent occasion. When colistin inhalation was discontinued in the first outbreak, the resistant isolates became undetectable within a short period of time and could not be cultured in routine microbiological examinations of sputum. This indicates that only the sensitive Fig. 2. The flowchart shows possible sources of transmission of colistin resistant Pseudomonas aeruginosa among the nine CF patients initially infected in 1995.

6 396 H.K. Johansen et al. / Journal of Cystic Fibrosis 7 (2008) strains persisted or dominated in the lungs when the antibiotic pressure disappeared. In the second outbreak, the resistant isolate was not cleared, probably because the resistant cluster now dominated the bacterial population in the lungs [32]. This is in accordance with the other CF study that reported on an outbreak of colistin resistant P. aeruginosa [11]. The geometric mean MIC of the sensitive non-mucoid and mucoid isolates towards colistin remained stable during the entire study period. This is in accordance with the findings in a randomized clinical trial comparing nebulized colistin with tobramycin in CF patients. The authors found that after 4 weeks of nebulized colistin the P. aeruginosa MICs remained unchanged [44]. In conclusion, the cohort isolation technique used in our CF centre involves a risk of spreading resistant clones within a cohort. Until now, two such epidemics have occurred, which in both cases were due to non-mucoid strains and did not lead to severe clinical problems for the patients [19]. However, individual isolation and avoidance of colistin use was necessary to stop the epidemic. Acknowledgement We thank Ulla Rydahl Johansen and Tina Wassermann for their technical assistance. References [1] Johansen HK, Høiby N. Seasonal onset of initial colonisation and chronic infection with Pseudomonas aeruginosa in patients with cystic fibrosis in Denmark. Thorax 1992;47: [2] Høiby N, Frederiksen B. Microbiology of cystic fibrosis. In: Hodson ME, Geddes DM, editors. Cystic Fibrosis. 2nd edition. London: Arnold; p [3] Høiby N, Frederiksen B, Pressler T. Eradication of early Pseudomonas aeruginosa infection. J Cyst Fibros 2005;4(Suppl 2): [4] Jensen T, Pedersen SS, Garne S, Heilmann C, Høiby N, Koch C. Colistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. 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