1 1 2 3 Angela Gomez-Simmondsa, Michelle Greenmana, Sean B. Sullivana, Joshua P. Tanner 4 a ...

34 Pages·2015·1.51 MB·English

JCM Accepted Manuscript Posted Online 15 April 2015
J. Clin. Microbiol. doi:10.1128/JCM.03455-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Population structure of Klebsiella pneumoniae causing bloodstream infections at a
2 New York City tertiary care hospital: diversification of multidrug-resistant isolates
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4 Angela Gomez-Simmonds a, Michelle Greenman a, Sean B. Sullivan a, Joshua P. Tanner
5 a, Madeleine G. Sowasha, Susan Whittierb, Anne-Catrin Uhlemann a,1 D
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7 a Department of Medicine, Division of Infectious Diseases, Columbia University d

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8 Medical Center, New York, NY 10032, United States
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9 b Department of Pathology and Cell Biology, Clinical Microbiology Laboratory, tt
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10 Columbia University Medical Center, New York, NY 10032, United States jc
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12 1Corresponding author: .o
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13 Anne-Catrin Uhlemann, Department of Medicine, Columbia University Medical o
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14 Center, New York, NY 10032, United States, Phone [phone], Fax 212-305- p
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15 5794, [email protected] 4
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18 Abstract:
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20 Despite the growing importance of carbapenem-resistant Klebsiella pneumoniae
21 (CRKP), the clonal relationships between CRKP and antibiotic-susceptible isolates
22 remain unclear. We compared the genetic diversity and clinical features of CRKP,
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23 3rd and/or 4th generation cephalosporin-resistant (Ceph-R), and susceptible K. w
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24 pneumoniae causing bloodstream infections at a tertiary care hospital in New York a
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25 City between January 2012 and July 2013. Drug susceptibilities were determined by d

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26 Vitek2. Isolates underwent multi-locus sequence typing and PCR-sequencing of the m

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27 wzi and blaKPC genes. Clinical and microbiologic data were extracted from patient tp
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28 records and correlated with molecular data. Among 223 patients, we identified 272 jc
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29 isolates. Of these, 194 were susceptible, 30 Ceph-R, and 48 CRKP and belonged to s
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30 144 sequence types (STs). Susceptible (127 STs) and Ceph-R (20 STs) isolates were r
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31 highly diverse. ST258 dominated CRKP strains (12 STs; 63% ST258). There was
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32 minimal overlap in STs between resistance groups. Bla (30%) was restricted to p
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33 ST258/wzi154, whereas bla (70%) included several wzi alleles. CRKP infections ,
KPC-2
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34 occurred more frequently in solid organ transplant (31%) and dialysis patients
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35 (17%). Mortality was overall high (28%) and highest in CRKP-infected patients
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36 (59%). In multivariable analyses, advanced age, comorbidities and the severity of s
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37 disease were significant predictors of 30-day mortality whereas the K. pneumoniae
38 susceptibility phenotype was not. Amongst CRKP infections we observed a
39 borderline significant association to increased mortality with ST258 and the wzi154
40 allele. Although the clonal spread of ST258 continues to contribute substantially to
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41 the dissemination of CRKP, non-ST258 strains appear to be evolving. Further
42 investigations into the mechanisms promoting CRKP diversification and the impact
43 of clonal background on outcomes are warranted.
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45 Introduction:
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47 Infections due to carbapenem-resistant Enterobacteriaceae have emerged as an
48 important public health problem over the past decade, and are now considered an
49 urgent antibiotic resistant threat by the Centers for Diseases Control (CDC), the
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50 category of highest concern (1). In the United States, carbapenem resistance among w
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51 Enterobacteriaceae is primarily attributable to the production of the Klebsiella a
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52 pneumoniae carbapenemase (KPC) (2, 3), which is plasmid-mediated and most d

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53 commonly encountered in K. pneumoniae in nosocomial settings (4, 5). Bloodstream m

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54 infections (BSI) caused by carbapenem-resistant K. pneumoniae (CRKP) are tp
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55 associated with particularly high mortality (6-8), with previous studies reporting jc
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56 hospital mortality rates of 40-70%, compared to 20-30% among matched patients s
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57 with bacteremias due to susceptible K. pneumoniae (4, 9). r
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58 A single clone, multi-locus sequence type (ST) 258, has been found to account
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59 for the majority of CRKP infections in the United States and was identified among p
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60 70% of isolates sent to the CDC for resistance testing from 1996 to 2008 (2). More ,

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61 recently, whole-genome sequencing revealed that ST258 comprises at least two
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62 distinct genetic lineages (10), highlighting the on-going evolution and adaptation of
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63 this strain. Its divergence was based on differences in a hypervariable region s
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64 encoding a K. pneumoniae capsule component as well as the presence of bla or
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65 bla . In a separate investigation, ST258 harbouring bla was exclusively
KPC-3 KPC-3
66 associated with wzi allele type wzi154, which corresponds to the novel capsule type
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67 C200 (11). This KPC-3-expressing strain was found to have decreased virulence in a
68 Galleria mellonella (waxworm) model compared to strains carrying bla .
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69 Despite this close association between bla variants and ST258 sublineage,
KPC
70 several studies have demonstrated that the Tn4401 transposon known to carry the
71 bla gene can be found on a variety of plasmids that are in turn transferrable
KPC
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72 between Enterobacteriaceae (12-14), although the nature of recipient strains has w
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73 not been well defined. While most carbapenem resistance in K. pneumoniae is a
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74 plasmid-mediated, alternative mechanisms such as the presence of porin mutations d

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75 in isolates that harbour extended-spectrum beta-lactamases (ESBL) have also been m

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76 described (15). However, given that most hospital epidemiologic investigations tp
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77 focus on identifying direct transmission events involving single clones, the jc
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78 contribution of plasmid-mediated horizontal gene transfer and other mechanisms to s
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79 the CRKP epidemic remains largely unknown. r
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80 To better understand the interaction between susceptible and drug-resistant
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81 K. pneumoniae populations, we investigated the molecular epidemiology of K. p
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82 pneumoniae BSI at a tertiary-care hospital in New York City. We assessed molecular ,

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83 variables in order to identify isolate characteristics, including sequence and capsule
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84 types, associated with the acquisition of multidrug resistance determinants. We also
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85 evaluated host factors associated with Ceph-R and CRKP infection, and the impact of s
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86 molecular factors on outcome. Understanding the isolate and host characteristics
87 that promote the emergence and transmission of CRKP among hospitalized patients
88 may inform the design of interventions that limit its dissemination.
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90 Materials and Methods:
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92 Study isolates and chart review: This study was reviewed and approved by the
93 Institutional Review Board of Columbia University Medical Center (CUMC). We
94 retrospectively identified and retrieved all K. pneumoniae isolates grown from blood
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95 cultures that were systematically stored by the microbiology laboratory from w
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96 January 2012 until July 2013. The microbiology laboratory processes all clinical a
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97 specimens obtained from the Columbia University hospital system, which includes d

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98 large academic adult and children’s teaching hospitals and a smaller community m

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99 hospital that serves as a referral centre for local skilled nursing facilities. Repeat tp
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100 isolates collected from a single patient within 14 days of the previous isolate were jc
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101 not available for analysis. We decided to include repeat isolates in our description of s
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102 clonal diversity to account for some degree of within-patient heterogeneity. r
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103 Isolates were retrospectively matched to patient medical records and clinical
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104 information was extracted by chart review. Detailed information on baseline p
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105 comorbidities and disease severity at the time of the positive bloodstream culture ,

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106 was used to calculate Charlson Comorbidity Index scores (CCIS) and Pitt
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107 bacteraemia scores (PBS), respectively. Clinical outcomes were defined as 1) the 30-
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108 day all-cause mortality from the date of bacteraemia and 2) mortality during the s
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109 index hospitalization. The presumed source of infection was determined using
110 CDC/NHSN surveillance definitions (16) and then grouped into the following
111 categories: central line-associated, respiratory tract, intraabdominal, soft tissue
112 (including wound), urinary tract, or other infection, or unknown source of infection.
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113 In addition, microbiology records were reviewed for results of antibiotic
114 susceptibility testing, which was performed according to routine microbiology
115 laboratory protocol using the Vitek2 automated system and additional E-testing as
116 needed (bioMerieux, Durham, NC). Susceptibility breakpoints were derived from the
117 Clinical and Laboratory Standards Institute guidelines (17). CRKP isolates were
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118 defined by nonsusceptibility to any carbapenem, and Ceph-R isolates were w
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119 nonsusceptible to ceftazidime, cefotaxime, ceftriaxone, or cefepime, in accordance a
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120 with infection control guidelines at our institution. Susceptible K. pneumoniae d

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121 isolates did not meet criteria for multidrug-resistant (MDR) phenotypes (Ceph-R or m

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122 CRKP). For patients with multiple isolates collected, only the initial isolate tp
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123 susceptibility profile was considered when assessing clinical factors and outcomes, jc
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124 and is reported in detail. s
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125 r
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126 Sequence typing: Genetic relatedness among K. pneumoniae bloodstream isolates
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127 was determined using multilocus sequence typing (MLST) as previously described p
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128 (18). Sequencing of the wzi gene locus was also performed on select isolates, ,

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129 including all Ceph-R and CRKP isolates, and susceptible isolates that shared a ST
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130 with Ceph-R or CRKP isolates (18). wzi sequencing has been shown by previous
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131 studies to be a molecular method for rapid capsule serotyping and as a proxy for K- s
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132 typing (11). Unique allele and sequence type numbers were requested from the
133 Institut Pasteur for clones that had not been previously reported.
134 All K. pneumoniae bloodstream isolates were screened for the presence of
135 carbapenem resistance-conferring genes, including bla , bla , bla , and bla ,
KPC VIM IMP NDM
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136 by PCR amplification using previously established primers and PCR parameters (19).
137 For isolates found to harbour bla , amplification followed by bi-directional DNA
KPC
138 sequencing of the bla PCR product was carried out in order to differentiate
KPC
139 among bla alleles (20).
KPC-
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141 Statistical analysis: Clinical parameters were described as frequencies or using the w
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142 mean ± standard deviation and the median for normally and non-normally a
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143 distributed variables, respectively, as appropriate. For patients with multiple d

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144 positive blood cultures, only the first available isolate was used in the assessment of m

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145 clinical variables and outcomes. Univariable analyses were used to identify clinical tp
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146 and isolate characteristics associated with MDR infection and outcome using χ2 or jc
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147 Fisher’s exact test for categorical variables or ANOVA or Kruskal-Wallis test for s
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148 continuously distributed variables as appropriate. To further analyze factors r
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149 associated with 30-day mortality, clinical variables with p-value of <0.1 were
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150 selected for inclusion in a parsimonious multivariable model using backward step- p
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151 wise selection, to which a categorical resistance phenotype variable was added a ,

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152 priori. Similar multivariable models were constructed to assess the relationship
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153 between a priori selected molecular variables and 30-day mortality. In all analyses,
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154 p-values of <0.05 were considered statistically significant. Data were analysed using s
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155 SAS 9.3 (SAS Institute Inc., Cary, NC).
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157 Results:
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159 Clinical characteristics of patients: During the 18-months study period, 223
160 patients with K. pneumoniae bloodstream infections were identified. Multiple
161 isolates were collected for 29 patients, resulting in a total of 272 K. pneumoniae
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162 blood isolates. Of the 223 patients, 170 had susceptible, 24 had Ceph-R, and 29 had w
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163 carbapenem-resistant K. pneumoniae infections based on Vitek testing of their initial a
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164 bloodstream isolate. Patient demographic and clinical characteristics are delineated d

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165 in Tables 1 and 2. Across all three groups, patients were noted to be elderly (median m

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166 age 62), have multiple comorbid conditions (30% had a CCIS ≥4), and have high tp
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167 rates of previous hospitalization (61% within the previous 6 months). jc
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168 In univariable analyses differences between the three groups were seen in s
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169 underlying comorbidities (Table 1) and timing and risk factors for BSI (Table 2). r
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170 Patients who developed Ceph-R or CRKP BSI were often chronically ill (42% had a
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171 CCIS ≥4). A large proportion of these patients were solid organ transplant (SOT) p
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172 recipients (26%), and had end-stage renal disease (ESRD) requiring chronic dialysis ,

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173 (13%) or cirrhosis (13%). Many resided in long-term care facilities (26%), although
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174 these patients accounted for a higher proportion of Ceph-R infections (29%) than
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175 CRKP infections (24%). The source of infection did not significantly differ between s
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176 patient groups.
177 Solid organ transplant (SOT) recipients accounted for the largest proportion
178 of patients with CRKP infections (31%), followed by nursing home residents (24%)
179 and patients with end-stage renal disease (17%) or cirrhosis (14%). Among SOT
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180 recipients, CRKP infections were most common in liver transplant patients (56% of
181 CRKP BSI among SOT recipients) although at our centre liver transplantation is
182 performed only 30-40% more often than heart or lung and half as often as kidney
183 transplantation (21). All patients with CRKP BSI had been exposed to antibiotics
184 within the past 6 months, and only 28% had received carbapenems. Patients with
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185 susceptible and Ceph-R infections often had initial positive blood cultures drawn on w
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186 or soon after admission, CRKP bacteraemia tended to develop later in the hospital a
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187 course. Finally, at the time of their bacteraemia, patients with CRKP infections were d

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188 more likely to be severely ill (68% had PBS ≥4 on the date of the positive blood m

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189 culture and 79% were in an ICU) than their counterparts with susceptible or Ceph-R tp
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190 isolates. Recent invasive surgery, central venous catheterization, Foley catheter use, jc
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191 and mechanical ventilation were correspondingly common in this group and may s
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192 have served as additional risk factors for MDR infection. r
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193
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194 Population structure of K. pneumoniae bloodstream isolates: We performed p
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195 MLST on all 272 isolates and detected 144 different STs (Figure 1a). Overall, ST258 ,

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196 was the predominant clone and was detected in 32 isolates (12%), followed by ST20
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197 (n = 14), ST17 (n = 12), and ST 37 (n = 10). There were 13 STs comprising 4 or more
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198 isolates, and a unique ST was identified in 108 isolates. s
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199 We observed the highest number of STs among the 194 susceptible isolates
200 (Figure 1b). We identified 127 different STs and most of these were singletons (99
201 STs). ST20 was the most common susceptible K. pneumoniae strain with 12 isolates
202 (19%), followed by ST37 (n = 10) and ST45 (n = 6). Moreover, only 5 STs were
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