# Patent

## Title
Method of identifying A. baumannii with OXA-131-like drug resistance in diabetic patients

## Abstract

A method for identifying A. baumannii with OXA-131-like drug resistance in diabetic patients includes the steps of obtaining a sample from a patient; identifying an isolate as A. baumannii; screening the isolate for genes encoding an OXA-51-like enzyme; sequencing any of the genes encoding an OXA-51-like enzyme; and identifying the isolate as OXA-131-like when the sequence matches the sequence for OXA-90 (SEQ ID NO: 1), OXA-130 (SEQ ID NO: 2), OXA-131 (SEQ ID NO: 3), or OXA-132 (SEQ ID NO: 4). The method may further include the step of identifying the ISAba1 sequence upstream from the gene encoding the OXA-131-like enzyme.

## Background
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN COMPUTER READABLE FORM
The Applicant hereby incorporate by reference the sequence listing contained in the ASCII text file titled 32087—00_rev_sequence_ST25.txt, created Apr. 24, 2012 and having 15.3 KB of data (16.0 KB on disk).
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods of identifying drug-resistant bacteria, and particularly to a method of identifying A. baumannii with OXA-131-like drug resistance in diabetic patients.
2. Description of the Related Art
Acinetobacter baumannii is an aerobic gram-negative species of bacteria that is resistant to most antibiotics. Infection by A. baumannii can result in pneumonia and infections of the urinary tract, the bloodstream, and other parts of the body. Left untreated, such infections can result in extended and debilitating convalescence, and may prove fatal. A. baumannii infects those with compromised immune systems, such as the wounded, the elderly, children, those with debilitating diseases, such as diabetes or other diseases or illnesses that might weaken the immune system. A. baumannii has been implicated as the source of nocosomial infections in hospitals and other treatment facilities, with the number of reported nocosomial infections linked to A. baumannii increasing in number in recent years.
The treatment of choice has traditionally been carbapenems (the carbapenems include meropenem, ertapenem, imipenem, doripenem, panapenem, and biapenem), since A. baumannii is resistant to many other antibiotics. The carbapenems are a class of beta-lactam antibiotics effective against a broad spectrum of bacterial infection and resistance to β-lactamases.
In recent years, however, many strains of A. baumannii have shown resistance to the carbapenems. Such resistance results from genes encoding carbapenemases, which have most commonly either been molecular class B metallo-β-lactamases, or molecular class D OXA β-lactamases (so-named for resistance to oxacillin and related compounds). The OXA β-lactamases exhibiting resistance to carbapenems have generally fallen into one of four subgroups: OXA-23-like, OXA-24-like, OXA-58-like, and OXA-51-like. The blaOXA-51-like gene is found in all strains of A. baumannii, and may be used as a marker for identifying A. baumannii. The OXA-51-like subgroup generally exhibits a low level of carbapenem resistance, but when the ISAba1 sequence is upstream of the gene encoding the enzyme, the OXA-51-like enzyme is overexpressed and the gene is more likely to replicate. It is thought that the ISAba1 sequence may act as a promoter when it is upstream from the gene encoding the OXA-51-like enzyme, signaling the start of the gene for messenger RNA to transcribe when expressing the enzyme. As used herein, the term “OXA-51-like” refers to a subgroup of OXA enzymes that includes a group of enzymes with OXA numbers 51, 64, 65, 66, 67, 68, 69, 70, 71, 75, 76, 77, 83, 84, 86, 87, 88, 89, 91, 92, 94 and 95. OXA enzymes may or may not have similar structure, and many of the genes encoding the enzymes may have no more than 20% homology in their sequence. β-lactamases are thought to confer drug resistance to carbapenemases by hydrolysing (or hydrolyzing) and opening the β-lactam ring, rendering the carbapenem ineffectual.
The present inventor has identified four new strains of A. baumannii with novel blaOXA-51-like genes, which encode four enzymes that the inventor refers to as OXA-131-like, the OXA-131 subgroup including OXA-90 (SEQ ID NO: 5), OXA-130 (SEQ ID NO: 6), OXA-131 (SEQ ID NO: 7), and OXA-132 (SEQ ID NO: 8). The OXA-90 gene has the sequence of nucleotides shown in SEQ ID NO: 1 in the attached Sequence Listing. The OXA-130 gene has the sequence of nucleotides shown in SEQ ID NO: 2 in the attached Sequence Listing. The OXA-131 gene has the sequence of nucleotides shown in SEQ ID NO: 3 in the attached Sequence Listing. The OXA-132 gene has the sequence of nucleotides shown in SEQ ID NO: 4 in the attached Sequence Listing.
Each enzyme encodes a β-lactamase that exhibits some degree of carbapenemase activity, and is also resistant to several other antibiotics. Because Infection by A. baumannii may lead to prolonged convalescence or may prove fatal, it is desirable is identify the strain of A. baumannii quickly in order to make an informed selection of an antibiotic for treatment of the infection.
Thus, a method of identifying A. baumannii with OXA-131-like drug resistance in diabetic patients solving the aforementioned problems is desired.
SUMMARY OF THE INVENTION
A method for identifying A. baumannii with OXA-131-like drug resistance in diabetic patients includes the steps of obtaining a sample from a patient; identifying an isolate as A. baumannii; screening the isolate for genes encoding an OXA-51-like enzyme; sequencing any of the genes encoding an OXA-51-like enzyme; and identifying the isolate as OXA-131-like when the sequence matches the sequence for OXA-90 (SEQ ID NO: 1), OXA-130 (SEQ ID NO: 2), OXA-131 (SEQ ID NO: 3), or OXA-132 (SEQ ID NO: 4). The method may further include the step of identifying the ISAba1 sequence upstream from the gene encoding the OXA-131-like enzyme.
These and other features of the present invention will become readily apparent upon further review of the following specification.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A method for identifying A. baumannii with OXA-131-like drug resistance in diabetic patients includes the steps of obtaining a sample from a patient; identifying an isolate as A. baumannii; screening the isolate for genes encoding an OXA-51-like enzyme; sequencing any of the genes encoding an OXA-51-like enzyme; and identifying the isolate as OXA-131-like when the sequence matches the sequence for OXA-90 (SEQ ID NO: 1), OXA-130 (SEQ ID NO: 2), OXA-131 (SEQ ID NO: 3), or OXA-132 (SEQ ID NO: 4). The method may further include the step of identifying the ISAba1 sequence upstream from the gene encoding the OXA-131-like enzyme.
In addition to the nucleotide sequences in the attached Sequence Listing, the amino acid sequences for each enzyme are also provided. The OXA-90 enzyme has the sequence of amino acids shown in SEQ ID NO: 5 in the attached Sequence Listing (i.e., SEQ ID NO: 1 is a nucleotide sequence of a portion of a gene encoding the OXA-90 enzyme, which has the amino acid sequence of SEQ ID NO: 5; the enzyme may be expressed under certain circumstances, notably when the ISAba1 sequence is upstream from SEQ ID NO: 1). The OXA-130 enzyme has the sequence of amino acids shown in SEQ ID NO: 6 in the attached Sequence Listing. The OXA-131 enzyme has the sequence of amino acids shown in SEQ ID NO: 7 in the attached Sequence Listing. The OXA-132 enzyme has the sequence of amino acids shown in SEQ ID NO: 8 in the attached Sequence Listing.
As described in the article A. A. Alsultan, A. Hamouda, B. A. Evans, S. G. B. Amyes, “Acinetobacter baumannii. Emergence of Four Strains with Novel blaOXA-51-like Genes in Patients with Diabetes Mellitus”, Journal of Chemotherapy, Vol. 21, No. 3 (290-295), 2009, which is hereby incorporated by reference in its entirety, the present inventor identified the four new strains of A. baumannii with OXA-131-like drug resistance as a result of a study of clinical samples obtained from diabetic patients at four hospitals and twenty medical centers in Saudi Arabia. All of the patients had diabetes mellitus and suffered from deep-sited infections.
Twenty isolates were identified phenotypically as A. calcoceticus/A. baumannii complex by API 20 NE (BioMerieux, Marcy L'Etoile, France). The isolates were further identified to the genomic level by restrictive polymorphism in the 16S-23S rRNA intragenic region. DNA from each strain was obtained by emulsifying a single colony from overnight growth, sequences of primer 1 (5′-TTG TAC ACA CCG CCC GTC A-′3) (SEQ ID NO: 9) and primer 2 (5′-GGT ACT TAG ATG TTT CAG TTC-′3) (SEQ ID NO: 10) were used to amplify a 975 bp section of the gene using PCR, and the amplification products were analyzed by gel electrophoresis.
The twenty isolates were screened for genes encoding OXA-51-like enzymes using multiplex PCR, with the primer pairs (5′-TAA TGC TTT GAT CGG CCT TG-′3) (SEQ ID NO: 11) and (5′-TGG ATT GCA CTT CAT CTT GG-′3) (SEQ ID NO: 12) being used to amplify a 353 bp segment of the blaOXA-51-like gene. PCR amplifications were also performed with the primers OXA69A (5′-CTA ATA ATT GAT CTA CTC AAG-′3) (SEQ ID NO: 13) and OXA-69B (5′-CCA GTG GAT GGA TGG ATA GAT TAT C-′3) (SEQ ID NO: 14) to obtain a 975 bp segment that contained the coding sequence of the entire blaOXA-51-like gene. Isolates producing a band of more than about 2000 bp resulting from an insertion upstream of the blaOXA-51-like gene were re-analyzed by using the primers preABprom+ (5′-GAC CTG CAA AGA AGC GCT GC-′3) (SEQ ID NO: 15) to generate an 1189 bp product, which was then sequenced.
All isolates were typed by pulsed field gel electrophoresis (PGFE). All isolates were analyzed by BioNumerics software version 4.
Of the twenty isolates, nine strains contained four novel OXA-51-like enzymes [four with genes encoding OXA-131 (SEQ ID NO: 3), one with a gene encoding OXA-130 (SEQ ID NO: 2), two with genes encoding OXA-90 (SEQ ID NO: 1), and two with genes encoding OXA-132 (SEQ ID NO: 4)], the remaining isolates containing other previously known OXA-enzymes. Of the four newly identified OXA-51-like enzymes, only the gene encoding the OXA-131 enzyme (SEQ ID NO: 3) also was found with the ISAba1 sequence upstream from the gene encoding the OXA-enzyme.
All twenty isolates were further tested for MICs (minimum inhibitory concentrations) for susceptibility to several known antibiotics using the British Society for Antimicrobial Chemotherapy guidelines. The results are summarized in Table I. It will be seen that all twenty isolates were highly resistant to ertapenem, oxacillin, ampicillin, sulbactam, chloramphphenicol, and tetracycline. The majority was also resistant to ciprofloxacin, gentamicin, piperacillin/tazobactam, amoxacillin/clavulanate, nalidixic acid, rifampicin, and ofloxacin. All isolates were susceptible to imipenem and colistin, and all but four isolates (which had low-level resistance) were susceptible to meropenem.







TABLE I







Antimicrobial susceptibility in 20 isolates











No. of resistant
MIC range
%


Antibiotic
isolates
(mg/L)
resistance













Imipenem
0
0.25-2   
0


Meropenem
4
4-8 
20


Ertapenem
20
4-64
100


Ciprofloxacin
9
1-16
45


Gentamicin
4
 32->128
20


Oxacillin
20
>128
100


Pipericillin/tazobactam
11
32-128
55


Ampicillin
20
 16->128
100


Amoxicillin/clavulanate
18
 16->128
90


Nalidixic Acid
15
 8->128
75


Colistin
0
0.25-0.5 
0


Rifampicin
16
4-32
80


Sulbactam
20
 64->128
100


Ofloxacin
14
1-32
70


Chloramphenicol
20
>128
100


Tetracycline
20
 2->128
100









It will be noted that all samples were from diabetic patients. However, the four OXA-131-like enzymes have not been previously identified in Acinetobacter baumannii isolates obtained from the general population. This suggests that these four new strains of Acinetobacter baumannii may evidence the emergence of drug-resistant Acinetobacter baumannii strains that pose a threat unique to the diabetic patient.
Diabetic patients are ten times more likely to develop Acinetobacter baumannii infections than the remainder of the population. Carbapenems are one of the very few antibiotics that effectively treat infections caused by this organism. Carbapenem-resistant strains of Acinetobacter baumannii can cause serious complications in diabetic patients.
The above identifies a new risk factor in diabetics. Emergence of new strains of A. baumannii isolated from patients with diabetes mellitus could reach epidemic levels. The percentage of A. baumannii isolates from those patients having strains with OXA-51-like β-lactamases was found to be high (approximately 45%). OXA-51-like β-lactamases are found to significantly contribute to carbapenem resistance, and this resistance correlates both with the survival of A. baumannii in diabetics and perhaps also in the development of diabetes.
There are certain gene variants that predispose people to develop diabetes, and these variations play a significant role in the development of a large spectrum of effects, which may include infections with A. baumannii. Nine representative isolates of A. baumannii from patients with diabetes mellitus were found to possess common gene variants encoding β-lactamases designated as OXA-90 (SEQ ID NO: 1), OXA-130 (SEQ ID NO: 2), OXA-131 (SEQ ID NO: 3), and OXA-132 (SEQ ID NO: 4). Previously, all variations in amino acids in OXA-51-like enzymes were found to be outside of class D carbapenemase motifs, which is consistent with the four enzymes identified in the above study.
The isolates with a polymerase chain reaction (PCR) product larger than c. 2000 bp using the primers OXA-69A and OXA-69B produced an 1189 bp product with the second set of primers. The nucleotide sequences of these strains had the insertion sequence ISAba1 located seven base pairs upstream from the open reading frame of the blaOXA-131 gene, which was the most frequent blaOXA-51-like gene (approximately 44%), compared to blaOXA-130 (approximately 11%), blaOXA-132 (approximately 22%), and blaOXA-90 (approximately 22%). Insertion of the sequence ISAba1 is widespread in A. baumannii with more than 13 copies in one cell and, through mobilization, can act as a “moving switch” to upregulate expression of downstream genes. The presence of ISAba1 upstream of blaOXA-131-like indicates that it has a significant role in both the transmission and expression of these genes to diabetic patients.
The study suggests that carbapenem resistance in A. baumannii may emerge in diabetics in regions that have, up to now, managed to avoid the problem. The dissemination of the strains of A. baumannii carrying the OXA-131 β-lactamase, with the insertion sequence ISAba1 upstream of the encoding gene, appears to threaten diabetics and may increase the risk due to the upregulation of blaOXA-131 gene expression.
Thus, in order to predict complications in patients with diabetes mellitus, the patients are tested for the existence of A. baumannii harboring the blaOXA-131 gene, with the insertion sequence ISAba1 being found upstream of the encoding gene. Testing whether a patient has A. baumannii harboring the blaOXA-131 gene with the insertion sequence ISAba1 upstream of the encoding gene may be an essential step in monitoring their carbapenem resistance phenotype and may assist in preventing their spread in diabetics, as well as allowing time for the individual patient to receive preventive care before the emergence of infectious symptoms.
It is to be understood that the present invention is not limited to the embodiment described above, but encompasses any and all embodiments within the scope of the following claims.

## Claims

1. A method for identifying an isolate as a member of a subgroup of A. baumannii with antibiotic drug resistance, comprising the steps of:
a) obtaining a sample from a patient;
b) identifying an isolate from the sample as A. baumannii; 
c) screening the isolate for genes encoding an OXA-51-like enzyme;
d) sequencing any genes encoding an OXA-51-like enzyme; and
e) identifying the isolate as a member of the subgroup of A. baumannii having antibiotic drug resistance when the sequence of the gene encoding an OXA-51-like enzyme determined in d) is OXA-90 (SEQ ID NO: 1), OXA-130 (SEQ ID NO: 2), OXA-131 (SEQ ID NO: 3), or OXA-132 (SEQ ID NO: 4).

2. The method for identifying a subgroup of A. baumannii with antibiotic drug resistance as recited in claim 1, further comprising the step of identifying the ISAba1 sequence upstream from the gene encoding the enzyme of the member of the subgroup having antibiotic drug resistance, thereby confirming drug resistance due to the enzyme.