Identification of blaOXA-51-like, blaOXA-58, blaDIM-1 and blaVIM Carbapenemase 1 Genes in Hospital Enterobacteriaceae Isolates from Sierra Leone 2
3 Tomasz A. Leski,a Umaru Bangura,b David H. Jimmy,b,c Rashid Ansumana,b,d,e Stephen E. 4
Lizewski,a* Robert W. Li,f David A. Stenger,a Chris R. Taitt,a and Gary J. Voraa** 5 6 Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, 7 District of Columbia, USAa; Mercy Hospital Research Laboratory, Kulanda Town, Bo, Sierra 8 Leoneb; Chemistry Departmentc and Institute of Environmental Management and Quality 9 Control, Njala University, Njala, Sierra Leoned; Liverpool School of Tropical Medicine, 10 University of Liverpool, Liverpool, UKe; and Bovine Functional Genomics Laboratory, Animal 11 and Natural Resources Institute, United States Department of Agriculture, Beltsville, Maryland, 12 USAf 13 *Present address: Infectious Diseases Research Directorate, Naval Medical Research Center, 14 Silver Spring, Maryland, USA 15 **Correspondence: Gary J. Vora, Center for Bio/Molecular Science and Engineering, Naval 16 Research Laboratory, 4555 Overlook Avenue – SW, Bldg. 30 / Code 6910, Washington, DC 17 20375, USA. Tel: 202.767.0394, Fax: 202.767.9594, Email: [email protected] 18 19 Keywords: carbapenemase, carbapenem-hydrolyzing class D β-lactamase, Enterobacteriaceae, 20 metallo-β-lactamase, multidrug resistance, molecular epidemiology 21 22 Article summary line: Carbapenemase genes in Sierra Leone 23
ABSTRACT 24 We describe the results of a molecular epidemiological survey of 15 carbapenemase-encoding 25 genes from a recent collection of clinical isolates from Mercy Hospital in Bo, Sierra Leone. The 26 most salient findings revealed that (i) 60% of the isolates harbored multiple carbapenemase 27 genes, (ii) the blaDIM-1 gene that has only been reported in The Netherlands is also circulating in 28 this environment and (iii) blaOXA-51-like and blaOXA-58 genes, which were thought to reside 29 exclusively in Acinetobacter species, can also be found in members of the Enterobacteriaceae. 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Carbapenem antibiotics are currently the most potent β-lactam antibiotics clinically 48 available and are used as a last resort to treat infections caused by multidrug resistant Gram-49 negative pathogens. The significance and usage of this family of antibiotics has risen 50 dramatically over the last decade, especially in hospital settings, due to the global spread and 51 increasing prevalence of bacterial pathogens that harbor extended spectrum β-lactamase genes 52 that confer resistance to all β-lactam compounds except carbapenems (1). Not surprisingly, the 53 positive selection pressure provided by the increased usage of carbapenems has resulted in the 54 emergence and expansion of carbapenem-resistance in a number of common nosocomial 55 pathogenic species (1). 56
Carbapenem resistance is mostly mediated by β-lactamase enzymes that are capable of 57 hydrolyzing carbapenem compounds (carbapenemases) and often differ in host microorganism 58 range, substrate specificity and β-lactamase inhibitor sensitivity (2, 3). Furthermore, 59 carbapenemases are frequently found in pathogenic strains that contain additional genetic 60 determinants that confer resistance to aminoglycosides, tetracyclines, β-lactams and 61 fluoroquinolones and can result in intractable infections with high mortality rates (4, 5). The 62 spread of Ambler class A, B and D carbapenemase genes, which were only rarely encountered 63 less than two decades ago, have been facilitated by conjugative plasmids, transposons and 64 integrons to such an extent that carbapenemase genes have now been reported worldwide (2). 65 Despite this fact and their clinical impact, the true incidence and prevalence of carbapenem 66 resistance and carbapenemase genes are still unknown as many countries do not track and report 67 antibiotic resistance rates and this is particularly true in many African countries (1). 68
To better understand the level, distribution and evolution of multidrug resistance in 69 environments that currently lack a national antibiotic stewardship policy, we initiated a 70
molecular epidemiology surveillance program at Mercy Hospital in Bo, Sierra Leone. The 71 preliminary evidence, obtained using the Antimicrobial Resistance Determinant Microarray as a 72 screening tool (6), indicated the presence of class B metallo-β-lactamase (MBL) and class D 73 carbapenem hydrolyzing β-lactamase (CHDL) genes in this environment. In this study, we 74 utilized PCR and DNA sequencing to target 15 carbapenemase genes (blaOXA-23, blaOXA-24, 75 blaOXA-48, blaOXA-51-like, blaOXA-58-like, blaAIM, blaBIC, blaDIM, blaGIM, blaIMP, blaKPC, blaNDM, blaSIM, 76 blaSMP, blaVIM) from a recent collection of Mercy Hospital isolates to better understand the 77 carbapenemase content of actively circulating strains. Ethical approval was obtained from the 78 Njala University Institutional Review Board. All clinical samples were obtained from Mercy 79 Hospital as pre-existing diagnostic specimens that had been stripped of all identifiers. The Gram-80 negative clinical isolates analyzed (n=20) were collected from November 2010 to April 2011 on 81 agar media and then identified based on the PCR amplification and sequencing of a 16S rRNA 82 gene amplicon that spanned the V3 and V4 variable regions (7) and the rpoB and/or gyrA gene as 83 previously described (8, 9). For a preliminary identification, the obtained 16S rDNA sequences 84 were used to classify each isolate using the Naïve Bayesian Classifier available through the 85 Ribosomal Database Project (Release 10) (http://rdp.cme.msu.edu/classifier/classifier.jsp) with ≥ 86 80% confidence as the identification threshold (10). This analysis identified 70% of the isolates 87 as members of the Enterobacteriaceae with the remaining isolates belonging to the 88 Pseudomonadaceae, Burkholderiaceae or Comamonadaceae. Secondary identifications based on 89 rpoB or gyrA gene sequencing not only confirmed the 16S rDNA identifications but also 90 provided genus and species level resolution to reveal members of well known nosocomial 91 pathogens (Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae). In addition, the 92 genomic DNA from one strain (SL-1) was subjected to low pass whole genome sequencing using 93
an Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA) and the assembled and annotated 94 draft sequences were mined for carbapenemase genes and their flanking regions. 95
The targeted PCR analyses were performed using previously described primers and 96 conditions (11, 12) and selected amplicons were sequenced for allele confirmation. Importantly, 97 11 of the 15 targeted carbapenemase genes were not detected in any of the isolates tested 98 (blaOXA-23, blaOXA-24, blaOXA-48, blaAIM, blaBIC, blaGIM, blaIMP, blaKPC, blaNDM, blaSIM, blaSMP). 99 However, the remaining targeted Ambler class B (blaVIM and blaDIM-1) and D (blaOXA-51-like and 100 blaOXA-58-like) carbapenemase genes were detected and co-detected in a surprising number of 101 strains (Table 1). In fact, 60% of the tested isolates harbored multiple carbapenemase genes. 102
Perhaps even more surprising was the detection of the blaOXA-58-like and blaOXA-51-like 103 CHDL genes in 85% and 40% of the isolates, respectively, as they are thought to reside 104 exclusively in Acinetobacter species. While blaOXA-58-like genes are known to reside on 105 conjugative plasmids in Acinetobacter spp., the blaOXA-51-like genes were considered to be 106 exclusively chromosomally encoded, intrinsic oxacillinases of A. baumannii and are used for 107 species identification and strain typing by many investigators (2, 13, 14). However, a number of 108 recent reports indicate that the blaOXA-51-like genes have been mobilized and are spreading to other 109 Acinetobacter spp. by conjugative plasmids (15, 16). Our findings bolster this contention and 110 corroborate a recent meeting abstract that has described the presence of blaOXA-51-like and blaOXA-111 58-like genes in Klebsiella pneumoniae and Escherichia coli isolates, respectively (17). 112 Furthermore, an analysis of the SL-1 draft genome sequence identified this strain as an 113 Enterobacter cloacae isolate that harbored a complete blaOXA-58 open reading frame surrounded 114 by flanking sequences that were 100% identical to ISAba3 found in a number of Acinetobacter 115 plasmids (GenBank accession no. KC004135). Taken together, the results demonstrate the 116
presence of the blaOXA-58 and blaOXA-51-like carbapenemase genes in non-Acinetobacter Gram-117 negative genera and suggest that these genes should no longer be considered as genetic 118 determinants that can be used for the specific identification of Acinetobacter species. 119
The blaOXA-51-like genes were detected in eight isolates (Table 1). Previously published 120 primers [OXA-51-likeF and OXA-51-likeR (12); 5’OXA-51-like-all-F and 3’OXA-51-like-all-R 121 (18)] were used to amplify and sequence these genes for allelic identification and to determine 122 the presence of flanking ISAba1 sequences [using primers ISAba1F (18) and OXA-51-likeR 123 (12)] (18). The obtained sequences were screened against a database of >80 currently known 124 blaOXA-51-like genes and a total of five different blaOXA-51-like variants were found (Supplemental 125 Figure S1). Nearly full length sequences (94% of the entire gene) of the blaOXA-51-like variants 126 revealed the presence of OXA-64 (strain SL-1), OXA-65 (strain SL-5) and OXA-98 (strains SL-127 9, SL-13, SL-14 and SL-15). For two of the strains (SL-11 and SL-12), it was only possible to 128 amplify and sequence ~30% of the gene. Although greater sequence coverage is needed for 129 unambiguous identification of these variants, the obtained sequence information was sufficient to 130 determine that the blaOXA-51-like gene in SL-11 contained a novel combination of single nucleotide 131 polymorphisms that warranted its classification as a new variant of OXA-71. The same amount 132 of sequence information also revealed that the SL-12 blaOXA-51-like gene matched both OXA-71 133 and OXA-241. ISAba1 insertion sequences were detected in each of the blaOXA-51-like gene 134 containing isolates, however, they were not found flanking the blaOXA-51-like genes. Similar 135 analyses were performed to determine the allelic identity of the 16 blaOXA-58-like genes that were 136 detected in these isolates. Only two different sequence variants were identified, one present in 137 strains SL-1 and SL-11 and the other in all of the remaining positive samples. The variants 138 differed by two SNPs (Supplemental Figure S2), both of which generated silent mutations. The 139
deduced amino acid sequences for both variants were identical and matched exactly with OXA- 140 58. 141
In addition to the CHDL genes, two MBL genes were also identified among the tested 142 isolates. Sixty percent of the isolates carried identical blaVIM sequences (which based on the 143 amplicon sequence were consistent with blaVIM-2 or four other less frequently observed variants - 144 blaVIM-10, blaVIM-16, blaVIM17 and blaVIM-30) and 40% of the isolates were found to contain 145 blaDIM-1. While blaVIM (and especially blaVIM-2) is acknowledged as one of the most commonly 146 isolated MBLs in Africa and throughout the world (19-22), the presence of blaDIM-1 has been 147 reported only once before in a Pseudomonas stutzeri isolate from The Netherlands (23). Here, 148 blaDIM-1 was found in hospital isolates belonging to the families Enterobacteriaceae, 149 Pseudomonadaceae, Burkholderiaceae and Comamonadaceae. Importantly, the complete blaDIM-1 150 gene sequence was also found within the SL-1 draft genome and was found to be flanked by 151 sequences consistent with a type 1 integron (GenBank accession no. KC004136). 152
In this study, we have presented the first molecular epidemiological survey of 153 carbapenemase genes in West Africa. With respect to antimicrobial resistance determinants, this 154 is a wholly uncharacterized environment. It is also an especially challenging environment in that 155 this lack of surveillance coincides with easy over-the-counter access to the most recent 156 commercially available antibiotics, little to no usage oversight, low-level awareness of infection 157 control practices and the absence of a national antibiotic stewardship policy. To begin to 158 ascertain whether these factors may have played a role as the selection pressure that led to these 159 findings, we conducted a voluntary survey of 15 independent pharmacies and five hospital 160 dispensaries (including Mercy Hospital) to determine the availability of antimicrobial 161 compounds in Bo, Sierra Leone. While none of the hospital dispensaries issued antibiotics 162
without a prescription, 87% of the pharmacies that had access to the same collection of 163 antibiotics as the hospital dispensaries acknowledged readily dispensing antimicrobial drugs 164 without a prescription. Interestingly, none of the survey participants acknowledged issuing the 165 following carbapenem antibiotics: imipenem/cilastatin, meropenem, ertapenem and doripenem; 166 and the availability and use of carbapenem antibiotics have not previously been reported for this 167 region. However, β-lactam antibiotics such as amoxicillin, ampicillin, cloxacillin, and to a lesser 168 extent penicillin, cefuroxime and ceftriaxone were readily issued (as were antibiotics from other 169 classes such as chloramphenicol, ciprofloxacin, clarithromycin, co-trimoxazole, doxycycline, 170 erythromycin, metronidazole, gentamicin, rifampin, tetracycline, ofloxacin and nitrofurantoin). 171 Overall, the survey suggested that antibiotics in general are available and in use for self-172 treatment without a prescription or any professional supervision. Thus, while the selection 173 pressure for the maintenance of the carbapenemase genes identified in this study does not appear 174 to be due to the indiscriminate use of carbapenem antibiotics per se, it is possible that these 175 alleles are maintained in this population due to the use of β-lactam antibiotics or their genetic 176 linkage to other elements that may provide a more direct selective advantage. Furthermore, 177 similar queries of antibiotic usage for veterinary medicine and food animal production have not 178 been conducted in this environment and thus cannot be dismissed as the source for such selection 179 pressure. 180
The serious implications of the dissemination of carbapenemase genes has led to the 181 assertion that the early identification of strains containing these molecular determinants is 182 necessary for effective infection control measures and informed therapeutic options (4, 24). 183 Although it cannot be presumed that the presence of these carbapenemase genes within different 184 genetic backgrounds will result in high resistance to carbapenem antibiotics (13, 25, 26), these 185
findings clearly demonstrate a broadening reservoir for these resistance determinants. Taken 186 together and considering the needs, resources and level of existing characterization of this 187 environment, our findings warrant the continued use of molecular tools to better understand the 188 scope, severity and evolution of carbapenemase gene circulation and multidrug resistance in this 189 part of the world. 190 191 ACKNOWLEDGMENTS 192 There are no conflicts of interest to declare. 193
This work was supported in part by the Joint Science and Technology Office, Defense 194 Threat Reduction Agency (D.A.S.) and by the Office of Naval Research via U.S. Naval Research 195 Laboratory core funds (G.J.V.). The opinions and assertions contained herein are those of the 196 authors and are not to be construed as those of the U.S. Navy, military service at large or U.S. 197 Government. 198 199 REFERENCES 200 1. Nordmann, P., L. Dortet, and L. Poirel. 2012. Carbapenem resistance in 201
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SL-21 Burkholderia (100) no amplification - 58 - -
SL-22 Pseudomonadaceae (100) no amplification - 58 + + a Derived from 16S rDNA gene sequencing. All identifications are Family- or Genus-level identifications. Numbers in parentheses indicate the identification confidence level. b Derived from rpoB gene sequencing using the methods of Mollet et al (8). All identifications are Genus- and Species-level identifications. Numbers in parentheses indicate the percent sequence identity to reference sequences. c Derived from gyrA gene sequencing using the methods of Tayeb et al (9). All identifications are Genus- and Species-level identifications. Numbers in parentheses indicate the percent sequence identity to reference sequences.
d Numbers indicate the identified OXA-51-like variant. The variants were identified using 94% of the complete gene sequence in all cases except for SL-11 and SL-12 for which ~30% of the gene sequences were obtained.
e Numbers indicate the identified OXA-58-like variant. The variants were identified using 56% of the complete gene sequence in all cases.
f Obtained amplicon was not sequenced. All other amplicons lacking this designation were confirmed by DNA sequencing. g Identified as a new variant of OXA-71.
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