A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis.

OBJECTIVES: Staphylococcus aureus (S. aureus) is the most commonly implicated organism in septic arthritis, a condition that may be highly destructive to articular cartilage. Previous studies investigating laboratory and clinical strains of S. aureus have demonstrated that potent toxins induced significant chondrocyte death, although the precise toxin or toxins that were involved was unknown. In this study, we used isogenic S. aureus mutants to assess the influence of alpha (Hla)-, beta (Hlb)-, and gamma (Hlg)-haemolysins, toxins considered important for the destruction of host tissue, on in situ bovine chondrocyte viability. METHODS: Bovine cartilage explants were cultured with isogenic S. aureus mutants and/or their culture supernatants. Chondrocyte viability was then assessed within defined regions of interest in the axial and coronal plane following live- and dead-cell imaging using the fluorescent probes 5-chloromethylfluorescein diacetate and propidium iodide, respectively, and confocal laser-scanning microscopy. RESULTS: Hla-producing mutants caused substantial chondrocyte death compared with the toxin-deficient control (Hla-Hlb-Hlg-), whilst mutants producing Hlb and Hlg in the absence of Hla induced minimal chondrocyte death. Coronal studies established that Hla-induced chondrocyte death started in the superficial zone of cartilage and spread to deeper layers, whereas Hlb and Hlg toxins were without significant effect. CONCLUSION: This study identified Hla as a highly potent S. aureus toxin that caused rapid chondrocyte death in bovine cartilage, with other toxins or metabolic products produced by the bacteria playing a minor role. The identification of Hla in mediating chondrocyte death may assist in the development of therapeutic strategies aimed at reducing the extent of cartilage damage during and after an episode of septic arthritis.Cite this article: I. D. M. Smith, K. M. Milto, C. J. Doherty, S. G. B. Amyes, A. H. R. W. Simpson, A. C. Hall. A potential key role for alpha-haemolysin of Staphylococcus aureus in mediating chondrocyte death in septic arthritis. Bone Joint Res 2018;7:457-467. DOI: 10.1302/2046-3758.77.BJR-2017-0165.R1.

Rapid in situ chondrocyte death induced by Staphylococcus aureus toxins in a bovine cartilage explant model of septic arthritis.

OBJECTIVE: To assess in situ chondrocyte viability following exposure to a laboratory strain and clinical isolates of Staphylococcus aureus. METHODS: Bovine cartilage explants were cultured in the presence of S. aureus 8325-4 (laboratory strain), clinical S. aureus isolates or non-infected culture medium of pH values 7.4, 6.4 and 5.4. All clinical isolates were isolated from the joint aspirates of patients presenting with S. aureus-induced septic arthritis (SA). At designated time points, in situ chondrocyte viability was assessed within defined regions-of-interest in the axial and coronal plane following live- and dead-cell image acquisition using the fluorescent probes 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), respectively, and confocal laser-scanning microscopy (CLSM). Cartilage water content, following S. aureus 8325-4 exposure, was obtained by measuring cartilage wet and dry weights. RESULTS: S. aureus 8325-4 and clinical S. aureus isolates rapidly reduced in situ chondrocyte viability (>45% chondrocyte death at 40 h). The increased acidity, observed during bacterial culture, had a minimal effect on chondrocyte viability. Chondrocyte death commenced within the superficial zone (SZ) and rapidly progressed to the deep zone (DZ). Simultaneous exposure of SZ and DZ chondrocytes to S. aureus 8325-4 toxins found SZ chondrocytes to be more susceptible to the toxins than DZ chondrocytes. Cartilage water content was not significantly altered compared to non-infected controls. CONCLUSIONS: Toxins released by S. aureus have a rapid and fatal action on in situ chondrocytes in this experimental model of SA. These data advocate the prompt and thorough removal of bacteria and their toxins during the treatment of SA.

Molecular characterisation and diversity in Enterobacter cloacae from Edinburgh and Egypt carrying bla(CTX-M-14) and bla(VIM-4) ß-lactamase genes.

The purpose of this study was to compare the carbapenemases and extended-spectrum ß-lactamases (ESBLs) associated with resistance, the genetic environment of these genes, and their location on plasmids among Enterobacter cloacae isolates from Edinburgh (UK) and Egypt. Nine E. cloacae isolates were obtained from Egypt (n=3) and Edinburgh (n=6). Antimicrobial susceptibility testing was performed by agar dilution. Molecular detection of carbapenemase genes, blaCTX-M-14 and the presence of integron structures was done by PCR and sequencing. Genotyping of the strains was performed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction. Plasmids were extracted to determine the location of the resistance genes. PCR sequencing revealed that all of the isolates carried the blaCTX-M-14 ESBL gene, whilst two isolates also carried the blaVIM-4 metallo-ß-lactamase gene. The blaCTX-M-14 genes in two isolates were associated with the ISEcp1 transposase. Analysis of the integrons found an intI1 integron associated with the complex ISCR1. The blaVIM-4 gene was identified in the form of a gene cassette within the class 1 integron, followed downstream by the resistance genes aacA7, dfrA1 and aadA2. PFGE revealed genetic relatedness among six isolates, whereas the others were diverse although related. Plasmid analysis revealed a single plasmid carrying both blaVIM-4 and blaCTX-M-14. In conclusion, the presence of insertion sequence ISEcp1 upstream of blaCTX-M-14 suggests its involvement in the expression and mobilisation of this gene. Linked carriage of blaVIM-4 and blaCTX-M-14 on the same plasmid in E. cloacae results in resistance to all ß-lactams and limits antibiotic treatment options.

High frequency of carbapenem-resistant Acinetobacter baumannii in patients with diabetes mellitus in Saudi Arabia.

Carbapenem-resistant Acinetobacter baumannii is becoming increasingly prevalent in patients with diabetes mellitus in the Middle East. We examined the relationship of these bacteria and their resistance mechanisms to the diabetic disease status of patients in Saudi Arabia. Susceptibilities of 271 isolates to carbapenems, tigecycline and colistin were determined, followed by detection of carbapenemase genes. A blaVIM gene was detected in ~95 % of isolates; blaOXA-23 and blaOXA-40 genes were also prevalent. Diabetic patients were significantly more likely to carry carbapenem-resistant isolates. Carbapenem-resistant A. baumannii is a serious problem in diabetic patients, and molecular detection of resistance mechanisms in these isolates is required.

Diversity in Acinetobacter baumannii isolates from paediatric cancer patients in Egypt.

Acinetobacter baumannii is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. It is increasingly reported as a multidrug-resistant organism, which is alarming because of its capability to resist all available classes of antibiotics including carbapenems. The aim of this study was to examine the genetic and epidemiological diversity of A. baumannii isolates from paediatric cancer patients in Egypt, by sequencing the intrinsic blaOXA -51-like gene, genotyping by pulsed-field gel electrophoresis and multi-locus sequence typing in addition to identifying the carbapenem-resistance mechanism. Results showed a large diversity within the isolates, with eight different blaOXA -51-like genes, seven novel sequence types and only 28% similarity by pulsed-field gel electrophoresis. All three acquired class-D carbapenemases (OXA-23, OXA-40 and OXA-58) were also identified among these strains correlating with resistance to carbapenems. In addition, we report the first identification of ISAba2 upstream of blaOXA -51-like contributing to high-level carbapenem resistance. This indicates the presence of several clones of A. baumannii in the hospitals and illustrates the large genetic and epidemiological diversity found in Egyptian strains.

Insertion sequence disruption of adeR and ciprofloxacin resistance caused by efflux pumps and gyrA and parC mutations in Acinetobacter baumannii.

Acinetobacter baumannii is a pathogenic bacterium responsible for a wide range of infections in immunocompromised patients. This study examined the role of insertional inactivation of the adeR gene and its effect on adeABC gene expression along with characterisation of the gyrA and parC mutations involved in ciprofloxacin resistance in three A. baumannii clinical isolates and their derivatives. Primers designed for the detection of adeSRABC detected the presence of ISAba16, which disrupted the adeR gene in strain Ab12M, and ISAba1, which disrupted the same gene in strains Ab18 and Ab209. A second copy of ISAba1 was detected upstream of the adeA gene in Ab209 leading to AdeABC pump expression. AdeIJK pump expression was seen in all of the isolates but was not as significant as AdeABC expression. Minimum inhibitory concentrations of ciprofloxacin were ≥256 mg/L for all of the isolates and a decrease of ≥8-fold was seen following addition of the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine. Fluorometric analysis also demonstrated active efflux, with upregulation of adeIJK and some genes of the adeABC operon in some strains. Sequencing of the quinolone resistance-determining region of the gyrA and parC genes revealed a Ser83→Leu change in the gyrA gene and a novel change of Ser80→Trp in the parC gene of Ab12, Ab12M and Ab209; in Ab18 there was a Ser80→Leu change in parC. This study shows the multifactorial contribution of different mechanisms in A. baumannii leading to ciprofloxacin resistance.

ISAba825 controls the expression of the chromosomal bla(OXA-51-like) and the plasmid borne bla(OXA-58) gene in clinical isolates of Acinetobacter baumannii isolated from the USA.

Four non-repetitive, clonally related (ST114), carbapenem-resistant Acinetobacter baumannii strains isolated in the USA were examined to understand the mechanisms of carbapenem resistance including screening for the presence of an insertion sequence upstream of the bla(OXA-51-like) gene, which could be involved in the control and expression of the antibiotic-resistance gene. We observed that the main mechanisms of carbapenem resistance were the result of the over-expression of the bla(OXA-58-like) and the bla(OXA-65) gene, both of which had the presence of ISAba825 upstream of the genes. The importance of this element was shown by isolating plasmid-cured isogenic strains that had lost the plasmid with the ISAba825-bla(OXA-58-like) genes but during that same process also lost the chromosomal ISAba825 element present upstream of the bla(OXA-65) gene. A 16-fold decrease in minimum inhibitory concentration of imipenem and an eight-fold decrease in the minimum inhibitory concentration of meropenem were seen in the isogenic strains that lost the plasmid. The study presents the first report of ISAba825 simultaneously governing the bla(OXA-65) gene and the bla(OXA-58-like) gene expression and also highlights the importance of this element in carbapenem-sensitive isogenic strains, which were once carbapenem resistant.

Role of ISAba1 and ISAba125 in governing the expression of blaADC in clinically relevant Acinetobacter baumannii strains resistant to cephalosporins.

Acinetobacter baumannii is a multi-resistant opportunistic nosocomial pathogen responsible for several outbreaks worldwide. It can cause several infections at various sites of the body. One of the main infections caused by this bacterium is ventilator-associated pneumonia in patients in intensive care units. Treating these infections is becoming difficult because of the high resistance to antimicrobial agents. This study compared the expression of the chromosomally encoded bla(ADC) gene in isolates having ISAba1, ISAba125 and no insertion upstream of the bla(ADC) gene in A. baumannii clinical isolates. It showed that the expression of bla(ADC) was six times greater when ISAba125 was present upstream of the gene in comparison with the constitutively expressed bla(ADC) gene with no insertion present upstream. The study indicated that ISAba125 has better promoters than ISAba1 and this is responsible for the overexpression of the bla(ADC) gene as they share considerable homology to the well-established Escherichia coli promoters. The -10 box of ISAba125 formed a fusion promoter with the -35 box of the bla(ADC) gene causing the bla(ADC) gene to be significantly overexpressed. The ability to upregulate the expression of bla(ADC) with the assistance of different insertion elements such as ISAba1 and ISAba125 has become an important factor in A. baumannii resistance to cephalosporins.

Klebsiella pneumoniae susceptibility to biocides and its association with cepA, qacΔE and qacE efflux pump genes and antibiotic resistance.

BACKGROUND: Although antiseptics are some of the most widely used antibacterials in hospitals, there is very little information on reduced susceptibility to these biocides and its relationship with resistance to antibiotics. AIM: To determine the relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE and qacE, as well as identifying the role of efflux pumps in conferring reduced susceptibility. METHODS: Susceptibility was assessed for five biocides: chlorhexidine, benzalkonium chloride, Trigene, MediHex-4, Mediscrub; and for 11 antibiotics against 64 isolates of Klebsiella pneumoniae. Susceptibility to all compounds was tested by the agar double dilution method (DDM) and the effect of efflux pumps on biocides determined by repeating the susceptibility studies in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The presence of the cepA, qacΔE and qacE genes was identified by polymerase chain reaction. FINDINGS: The bacteria were not widely antibiotic resistant though a few showed reduced susceptibility to cefoxitin, chloramphenicol and rifampicin and later-generation cephalosporins but not to carbapenems. Biocide susceptibility, tested by DDM, showed that 50, 49 and 53 strains had reduced susceptibility to chlorhexidine, Trigene and benzalkonium chloride, respectively. The antiseptic resistance genes cepA, qacΔE and qacE were found in 56, 34 and one isolates respectively and their effects as efflux pumps were determined by CCCP (10 mg/L), which decreased the minimum inhibitory concentrations (MICs) of chlorhexidine and Medihex-4 by 2-128-fold but had no impact on the MICs of benzalkonium chloride, Trigene and Mediscrub. CONCLUSION: There was a close link between carriage of efflux pump genes, cepA, qacΔE and qacE genes and reduced biocide susceptibility, but not antibiotic resistance, in K. pneumoniae clinical isolates.

Rapid acquisition of decreased carbapenem susceptibility in a strain of Klebsiella pneumoniae arising during meropenem therapy.

A strain of Klebsiella pneumoniae (K1) was isolated from a catheterized patient with a urinary tract infection. The patient was subsequently treated with meropenem, after which K. pneumoniae (K2) was once again isolated from the patient's urine. Susceptibility testing showed that strain K1 was fully susceptible to carbapenem antibiotics with the exception of ertapenem, to which it exhibited intermediate resistance, whilst K2 was resistant to ertapenem and meropenem. From pulsed-field gel electrophoresis profiling both strains exhibited identical banding patterns. Both contained CTX-M-15, OXA-1, SHV-1 and TEM-1 ß-lactamase genes following PCR analyses. Outer membrane protein analysis demonstrated that K1 and K2 lacked an OMP of c. 40 kDa, with an additional OMP of c. 36 kDa missing from K2. Mutation studies showed that the K2 OMP phenotype could be selected by single-step carbapenem-resistant mutants of K1. Expression of transcriptional activator ramA and efflux pump component gene acrA were up-regulated in both strains by RT-PCR. Neither strain expressed ompK35, but ompK36 was found in both. Sequence analysis revealed gene sequences of ompK35, ompK36 and ramR in both strains; notably, ramR contained a mutation resulting in a premature stop codon. Transconjugation studies demonstrated transfer of a plasmid into E. coli encoding the CTX-M-15, TEM-1 and OXA-1 ß-lactamases. We concluded that the carbapenem-resistant phenotype observed from this patient was attributable to a combination of CTX-M-15 ß-lactamase, up-regulated efflux and altered outer membrane permeability, and that K2 arose from K1 as a direct result of meropenem therapy.

First report of a novel extended-spectrum beta-lactamase KOXY-2 producing Klebsiella oxytoca that hydrolyses cefotaxime and ceftazidime.

Klebsiella oxytoca strains MU946294N and MB193997E were isolated from patients in Scotland. Strain MU946294N was resistant to pencillins, monbactams and cephalosporins. Isolate MB193997E displayed a ß-lactam resistance phenotype consistent with chromosomal ß-lactamase overproduction. No bla(TEM), bla(SHV) or bla(CTX-M) genes could be amplified in either strain; however, amplification by PCR was found with primers for the bla(OXY-2) gene. This ß-lactamase gene in MU946294N differed by one mutation from the all other bla(OXY) genes previously reported, with an amino acid substitution Alanine237 Threonine enhancing the binding of cefotaxime. Strain MB193997E showed mutations at positions 255 and 283, neither of which affect function. Based on rpoB and gyrA characterization, both strains were assigned to the KoII phylogenic group but they were completely dissimilar from each other by PFGE. This study is the first to report the substitution of Alanine to Threonine at position 237 in a OXY- 2 ß-lactamase and this enhances resistance to cefotaxime.

Effect of frameshift mutagen acriflavine on control of resistance genes in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogenic bacterium that often exhibits a multidrug-resistant phenotype causing infections at various sites of the body and increasingly leading to septicaemic shock. This study evaluated the role of acriflavine, a frameshift mutagen, on the movement of insertion sequence ISAba1 in clinical isolates of A. baumannii, with the focus on changes in expression levels of the bla(ADC) and bla(OXA-51-like) genes. Resistance profiles were assessed with consideration of ISAba1 acting as a promoter upstream of the bla(ADC) or bla(OXA-51-like) gene. ISAba1 movement was observed in the acriflavine mutants Ab153M and Ab1225M. Ab153M exhibited an increase in the MIC values of carbapenems and ceftazidime, with ISAba1 gained upstream of the bla(ADC) and bla(OXA-51-like) genes, correlating with an increase in gene expression. Reduced expression of the 17, 23 and 25 kDa outer-membrane proteins (OMPs) was also observed in Ab153M. There was a significant decrease in MIC values of carbapenems with the loss of ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes in strain Ab1225M, and a significant decrease in bla(OXA-51-like) gene expression and, to a lesser extent, in bla(ADC) expression. Ab1225M and a serially subcultured Ab1225 strain (Ab1225s) exhibited overexpression of the 17, 23, 25 and 27 kDa OMPs. There was a decrease in MIC values of the carbapenems and piperacillin/tazobactam but not of ceftazidime in Ab1225s, which had ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes. A significant decrease in bla(OXA-51-like) expression was observed in Ab1225s, whereas the expression of bla(ADC) was similar to that in the Ab1225 parental strain. The attenuation in this strain may be due to overexpression of OMPs and it is clear that, even if ISAba1 is present upstream of an antibiotic resistance gene, it may not necessarily contribute towards the overexpression of antibiotic resistance genes (bla(OXA-51-like) in Ab1225s). Movement of the IS element within the A. baumannii chromosome may be an important regulatory mechanism employed by the bacterium under particular stress conditions, and the ability to upregulate the expression of antibiotic resistance genes is likely to be an important factor in the pathogenicity of this bacterium.

Prevalence of transferable blaCTX-M-15 from hospital- and community-acquired Klebsiella pneumoniae isolates in Scotland.

OBJECTIVES: This study was performed to investigate the prevalence and genetic characteristics of transferable bla(CTX-M-15) from hospital- and community-acquired Klebsiella pneumoniae isolates in Scotland. METHODS: A total of 219 clinical isolates of K. pneumoniae collected in 2006 and 2007 at the Royal Infirmary of Edinburgh, Scotland, were tested for antimicrobial susceptibility by the agar double dilution method. PCR and sequencing were used to detect bla(CTX-M), bla(TEM), bla(SHV) and qnr genes. Clonality of the isolates was assessed by PFGE. RESULTS: Sixteen (7.3%) isolates were found to be producers of CTX-M-15 extended-spectrum ß-lactamases (ESBLs), of which two isolates (12.5%) were reported to be from patients with community-acquired infections. The ISEcp1 was detected by sequencing 48 nucleotides upstream of bla(CTX-M-15) in all isolates but one. A total of one to two plasmids, ranging in size from ~40 to 210 kb, were observed per strain. By a PCR-based replicon typing method, plasmids carrying bla(CTX-M-15) were assigned to IncFII or IncN types. Sequencing and PCR analysis revealed the presence of complex class 1 integrons in all isolates but one. Two isolates positive for class 1 integrons were positive for class 2 integrons as well. Five different clones of CTX-M-15-producing isolates were identified by PFGE. CONCLUSIONS: This work reports the emergence of hospital- and community-acquired CTX-M-type enzymes in the Edinburgh area of Scotland.

Retrospective study of an outbreak in a Kuwaiti hospital of multidrug-resistant Klebsiella pneumoniae possessing the new SHV-112 extended-spectrum beta-lactamase.

Patients infected with bacteria producing extendedspectrum beta-lactamases (ESBL) are at higher risk of mortality and morbidity. Several mutations in genes encoding SHV, tem and CTX-M beta-lactamases have been associated with ESBL activity. This paper describes a new SHV mutation in ESBL-producing strains of Klebsiella pneumoniae isolated in Kuwait. The study included 13 K. penumoniae strains isolated from patients admitted to the Amiri hospital of Kuwait. The production of ESBL in all strains was confirmed by Vitek system and E-test. All the ESBL genes were amplified by PCR and examined by DNA sequencing. All these ESBL-positive isolates were resistant to ceftazidime and cefotaxime. DNA sequencing revealed an A815G point mutation in the bla (SHV )gene causing an asparagine (AAT) to aspartic acid (GAT) mutation at position 253 of the enzyme. This new mutation was assigned the unique number SHV-112, and the Genebank accession number EU477409. This study reports a new mutation in the SHV gene in K. pneumoniae with ESBL capability. There could be other mutations still to be found in ESBL genes of K. pneumoniae in Kuwait and probably in other middle eastern countries, and researchers in the region should make use of molecular techniques to look for more novel mutations in ESBL-producing strains of K. pneumoniae.

Novel genetic context of multiple bla OXA-58 genes in Acinetobacter genospecies 3.

OBJECTIVES: The detection in Acinetobacter genospecies 3 isolates of OXA-type carbapenemases, resulting in reduced susceptibility to carbapenem antibiotics, is increasingly reported. We identified an Acinetobacter genospecies 3 isolate carrying the gene for OXA-58 and aimed to resolve the genetic environment surrounding the bla(OXA-58) gene. METHODS: Species identification was confirmed by 16S-23S rRNA restriction analysis. MICs of imipenem, meropenem and ertapenem were determined, and the isolate was screened by PCR for bla(OXA-23-like), bla(OXA-40-like), bla(OXA-51-like) and bla(OXA-58-like) genes. The sequence surrounding bla(OXA-58) was determined through amplification by inverse PCR and genome walking followed by sequencing. Genetic localization was investigated by Southern blotting. RESULTS: Isolate A164 was confirmed as belonging to Acinetobacter genospecies 3 and had reduced susceptibility to the carbapenems. The isolate was found to encode two bla(OXA-58) genes that may have been duplicated by the insertion sequence ISAba125, two copies of which were inserted into ISAba3 elements. The bla(OXA-58) genes appear to be plasmid borne. CONCLUSIONS: This is the first report of beta-lactamase duplication in Acinetobacter genospecies 3 and of gene duplication mediated by ISAba125.

Taguchi optimisation of a multiplex pneumococcal serotyping PCR and description of 11 novel serotyping primers.

Recently, a PCR-derived method for serotyping Streptococcus pneumoniae has been devised to substitute the conventional antiserum phenotypic method. The method initially used a multiplex PCR reaction, dividing the isolates into 6 different groups based on the detected PCR gel pattern. In order to optimise and refine this crucial step, the Taguchi technique was employed, which can evaluate the individual effect of six parameters (in this case: primers, MgCl2, nucleotide mix, polymerase and buffer), with only 18 experiments; varying the parameter levels in an orthogonal matrix which suppresses the interactions between them. With this method, clear and sharp bands were observed in 5 experiments out of the 18, while the PCR did not work reliably in the remaining cases. In addition, the PCR-based technique could be rendered more economic by the 10-fold lowering of the quantities of two primers. The modified reaction yielded identical results to those obtained with the original method. Furthermore, we have designed serotype-specific primers for 11 new serotypes. The most important ones are those that can distinguish the very closely related, but equally important serotypes 6A and 6B.

Acinetobacter baumannii: emergence of four strains with novel bla(OXA-51-like) genes in patients with diabetes mellitus.

UNLABELLED: Diabetic patients are 10 times more likely to develop Acinetobacter baumannii infections than the rest of the population. Carbapenems are considered one of the very few antibiotics left to treat infections caused by this organism. the aim of this work was to characterise A. baumannii strains isolated from diabetic patients and to investigate whether there is a relationship between certain strains and low-level-carbapenem resistance. METHODS: Clinical samples were collected from diabetic patients in hospitals throughout Saudi Arabia from December 2006 to April 2007. API 20 Ne, polymorphisms in the 16S-23S-rRNA intergenic region and the presence of a bla( OXA-51-like )gene were all used for identification. Susceptibility to antimicrobials was determined using agar dilution and disk diffusion methods. pulsed-field gel electrophoresis (pfGe) coupled with sequence analysis of the bla(OXA-51-like )genes were used for strain characterization. Polymerase chain reaction (pCR) and multiplex pCR were used to screen for the presence and location of ISAba1 elements and bla(OXA-23-like), bla(OXA-40-like), and bla(OXA-58-like )genes respectively. RESULTS: Twenty isolates were identified as A. baumannii and were all highly resistant to 38% of the antibiotics tested and the majority of isolates were also resistant to 50% of the remaining antibiotics. four strains had low-level meropenem resistance (MIC 4-8 mg/l). All isolates were sensitive to imipenem and colistin. Nine strains possessed four novel bla( OXA-51-like )genes encoding beta-lactamases designated OXA-90, OXA-130, OXA-131 and OXA-132, and four strains contained bla(OXA-131 )with ISAba1 upstream of the gene structure. PFGE showed five separate clusters of OXA-51-like enzymes and the dissemination of strains carrying the four novel enzymes was clonal. this study showed that new strains of A. baumannii characterised by their new bla(OXA-51-like )gene have emerged. No genes encoding OXA-23-like, OXA-40-like, or OXA-58-like beta-lactamases were found. Surveillance of A. baumannii harbouring the bla( OXA-131-like )gene may be an essential step in monitoring their carbapenem resistance phenotype and may assist in preventing their spread in diabetics.