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.

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.

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.

OXA-51-like beta-lactamases and their association with particular epidemic lineages of Acinetobacter baumannii.

Sixty diverse clinical Acinetobacter baumannii isolates of worldwide origin were assigned to sequence groups, based on a multiplex PCR for the ompA, csuE and bla(OXA-51-like) genes. The majority (77%) of isolates belonged to sequence groups 1 and 2 (SG1 and SG2), with sequence group 3 (SG3) and non-grouped isolates accounting for the remainder. The isolates were not closely related according to pulsed-field gel electrophoresis (PFGE), and the majority were sensitive to imipenem and meropenem. The construction of a linkage map of OXA-51-like beta-lactamase sequence relationships revealed two closely related clusters of enzymes, one focused around OXA-66 and the other around OXA-69. Isolates belonging to SG1 encoded an enzyme from the OXA-66 cluster, while those belonging to SG2 encoded an enzyme from the OXA-69 cluster. All SG3 isolates encoded OXA-71, which does not form part of a close enzyme grouping. Major multinational lineages accounted for a significant proportion of A. baumannii clinical isolates, and the evolution of the OXA-51-like enzymes appears to be an ongoing process.

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.

Carbapenem resistance in clinical isolates of Pseudomonas aeruginosa.

The objectives of this study were to identify the carbapenem resistance mechanisms of clinical Pseudomonas aeruginosa isolates. The strains resistant to imipenem had lost only the OprD protein, the isolates resistant to imipenem and meropenem had both loss of the OprD porin and reduced minimum inhibitory concentrations (MICs) in the presence of efflux pump inhibitors. In the isolates in which efflux had been identified (n=2) only 1 isolate had a mutation in the mexR gene corresponding to a glutamine to a stop codon change at amino acid 106. This has not been previously identified. There were no significant changes in the mexT genes. No mutations previously associated with the upregulation of the carbapenem efflux pumps in in vitro generated resistant isolates were identified in any of the clinical isolates. Therefore, the resistance mechanisms identified by development of carbapenem resistance in vitro are not sufficient to understand carbapenem resistance development in clinical isolates.

Characterization of extended-spectrum beta-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains.

Two hundred and fifty-one unique patient isolates of Klebsiella pneumoniae (123), Escherichia coli (114), Klebsiella oxytoca (7), Enterobacter cloacae (5) and Citrobacter freundii (2), flagged as extended-spectrum beta-lactamase (ESBL) positive by the Vitek system (GNS-526 card), were collected. These strains were isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of the Royal Infirmary of Edinburgh (RIE), Edinburgh, UK (and associated GP practices), Hairmyers Hospital, Glasgow, UK, and the Amiri and Farwania Hospitals, Kuwait. Of the 101 RIE strains tested, 15 E. coli strains were found to be ESBL negative by Etest ESBL strips. On retesting the 15 E. coli strains with the Vitek GNS-532 card, 14 were found to be ESBL negative, despite being originally flagged as ESBL positive. The remaining 236 ESBL-producing strains were also subjected to the double disc-diffusion (DDD) technique for the detection of ESBLs. Of these, two were false negatives by Etest ESBL test strips (using both cefotaxime and ceftazidime strips), and 38 were false negatives by the DDD method. The Etest false-negative ESBL-producing strains of K. pneumoniae were positive by DDD. Technically, the Vitek method was the least demanding method to perform, as it was an integral part of the routine susceptibility test card. Etest strips were reliable, but were the most expensive of all the techniques used. The DDD test, while relatively inexpensive, was technically subjective, and in our hands, seven of the ESBL-positive strains that were confirmed by the other two techniques were not detected. Despite the false-positive ESBL-producing E. coli strains, the Vitek susceptibility card with its integral ESBL test offers the clinical laboratory a valuable and quick option to screen for ESBL-producing Klebsiella spp. and E. coli as part of the routine laboratory methodology.

Linkage of ciprofloxacin resistance with a single genotypic cluster of Klebsiella pneumoniae.

The objective of this study was to examine the epidemiology of ciprofloxacin-resistant, extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains. Sixty-nine unique patient isolates of K. pneumoniae isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of The Royal Infirmary of Edinburgh and associated General Practices were identified and susceptibility testing was performed with the Vitek system. Strains flagged as ESBL-positive by the Vitek system were subjected to isoelectric focusing. The results suggested that all 69 isolates harboured at least one ESBL, which was later confirmed by polymerase chain reaction (PCR) with bla(TEM) and/or bla(SHV) primers. The purified PCR product was subjected to automated sequencing and the results were compared with the BLAST online search engine. Of the 69 isolates, 32 (46.4%) were found to be resistant to ciprofloxacin, 11 (15.9%) were intermediate and 26 (37.7%) were sensitive. To investigate the epidemiological relationship between the ciprofloxacin-resistant ESBL-positive strains, pulsed-field gel electrophoresis (PFGE) was performed. Rapidest software was used to calculate the genetic distance by the Nei distance method. PFGE analysis indicated that the clinical isolates belonged to four distinct genotype clusters (Groups A, B, C and D); each group or cluster was homogeneous or compact with respect to certain characteristics. Group A consisted of 25 isolates, group B of 3 isolates and Groups C and D of 2 isolates each. These results indicate that the spread of resistance is largely as a result of the dissemination of a single clonal strain. PCR was used to amplify the gyrA and parC genes from genomic DNA of the ciprofloxacin-resistant isolates. The amplified product was sent for analysis by automated DNA sequencing and the resulting DNA sequences were compared with the gyrA gene of K. pneumoniae. The sequencing results demonstrated that alteration of the GyrA subunit of DNA gyrase at amino acid 83 and/or amino acid 87 plays a central role in conferring high-level quinolone resistance in K. pneumoniae possessing ESBLs.

Molecular characterisation of bovine faecal Escherichia coli shows persistence of defined ampicillin resistant strains and the presence of class 1 integrons on an organic beef farm.

Antimicrobial use is heavily restricted on organic farms; however, few studies have been conducted to investigate the impact this has on the epidemiology of resistance in pathogenic and commensal bacteria. We investigated the persistence of antimicrobial resistant Escherichia coli within an organic beef herd over a period of 28 months. Faecal samples collected monthly from three calf cohorts and annually from adult cattle and environmental samples, were screened for the presence of ampicillin, apramycin and nalidixic acid resistant E. coli. The prevalence of ampicillin resistance ranged from 27.3 to 40.7% in the annual herd and environmental samplings (n=22-55) and was greater in the calf cohorts, with a peak cohort prevalence of >47% in all 3 years (n=16-18). Apramycin and nalidixic acid resistant E. coli were rare. Pulsed-field gel electrophoresis (PFGE) identified 10 main genotype groups within the herd, with evidence of strain transmission between different livestock groups, animal species and years. Multiple resistance was found in >44% of isolates tested, with ampicillin, neomycin, sulphamethoxazole and tetracycline carriage the commonest phenotype identified. PCR detected the presence of class 1 integrons in <5% of resistant isolates, 6/7 of which were of cattle origin. These data demonstrate that ampicillin resistant E. coli was common on the farm despite restricted antimicrobial use, although strain diversity was low. Persistence of defined genotype groups was observed between years, together with the transmission of resistant strains between different animal species on the farm.

The first steps towards fluoroquinolone resistance in Hungarian pneumococci.

The incidence of fluoroquinolone resistance among Hungarian routine laboratory Streptococcus pneumoniae isolates, collected in 2000-2002, in common with other European countries, was very low; only 5/304 strains (1.64%) were resistant to ciprofloxacin (MIC = 4 microg/ml), and the other fluoroquinolones showed full efficacy. However, we could identify the Lys-137-Asp amino acid change, caused by a point mutation in the QRDR of the parC gene, in five strains. Additionally, we observed a definite shift in the minimum inhibitory concentrations (MICs) of all fluoroquinolones towards higher values throughout the study period. These two findings, coupled with the increasing consumption figures of fluoroquinolones, suggest that pneumococcal resistance looks poised to develop in Hungary.

The sequences of seven class D beta-lactamases isolated from carbapenem-resistant Acinetobacter baumannii from four continents.

Carbapenem resistance associated with class D beta-lactamases is an increasing problem in Acinetobacter baumannii. Most enzymes of this class reported so far belong to two subgroups, 1 and 2; however, a novel class D carbapenemase (OXA-51) has been reported recently which shares 56% and < 63% amino-acid identity with subgroups 1 and 2, respectively, and which belongs to a third subgroup. This study describes a further seven novel subgroup 3 beta-lactamases in carbapenem-resistant A. baumannii isolates from four continents.

Characterisation of OXA-51, a novel class D carbapenemase found in genetically unrelated clinical strains of Acinetobacter baumannii from Argentina.

Acinetobacter baumannii is now one of the most frequently encountered nosocomial pathogens in intensive therapy units, and is renowned for being difficult to treat because of resistance to most antibiotics. Carbapenems are the remaining drugs of choice in many centres, but carbapenem resistance is now emerging in strains worldwide. Two subgroups of carbapenem-hydrolysing beta-lactamases, which differ in their amino-acid homology, have been found in some resistant strains. This report describes the emergence and characterisation of a novel carbapenemase (OXA-51) in genetically distinct carbapenem-resistant A. baumannii strains from Argentina. Enzyme kinetics and inhibitor studies were performed spectrophotometrically with purified beta-lactamase. Amplification of the gene was achieved with a two-step PCR method employing arbitrary partially degenerate and gene-specific primers. Transfer of imipenem resistance was attempted with the use of broth and membrane filter methods. Attempts to produce plasmid-cured variants were made in ethidium bromide curing experiments. OXA-51 was identified in two clones of A. baumannii, and was found to have < 63% amino-acid identity with subgroups 1 and 2. Enzyme kinetic studies confirmed that OXA-51 was a molecular class D enzyme with carbapenemase activity, and that it displayed the highest affinity for imipenem (Km value 11 microM). Sequence analysis of the gene identified distinct differences within conserved class D motifs when compared with subgroups 1 and 2. Attempts to transfer imipenem resistance and to determine a plasmid location for the gene failed. OXA-51 is the first of a new subgroup of carbapenemases to emerge in multiresistant clinical isolates of A. baumannii.

The relationship between serotypes and PFGE genotypes in isolates of Streptococcus pneumoniae from Hungary.

The relatedness of 112 penicillin-non-susceptible isolates of Streptococcus pneumoniae from Hungary was determined by pulsed-field gel electrophoresis (PFGE), serotyping and antibiotic susceptibility tests. The differences in PFGE patterns closely mirrored the changes in resistance. Some genotypes comprised multiple serotypes, and the genetic diversity among certain serotypes was considerable. Generally, serotyping alone was insufficient for epidemiological mapping of pneumococcal isolates. There was considerable serotype diversity, but the five most frequent international serotypes (6, 9, 14, 23, 19) were the most prevalent. In addition, the presence of some well-defined resistant international pneumococcal clones in the Hungarian population was identified.

Epidemiological analysis of carbapenem-sensitive and -resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa with decreased levels of meropenem susceptibility were identified in the Royal Infirmary Edinburgh in 2002. Within the affected group of patients, none had meropenem-resistant P. aeruginosa when they arrived in the intensive care unit (ICU). Seven isolates from the ICU were collected five months after the decreased susceptibility to meropenem was identified. In order to investigate if resistance was a problem in P. aeruginosa throughout Edinburgh, both in hospital- and community-acquired isolates, a prospective study was performed. The susceptibilities of 104 P. aeruginosa to imipenem, meropenem, ceftazidime, piperacillin/tazobactam and ciprofloxacin were investigated. Meropenem had the highest activity against these isolates and the lowest MIC(90) (2 mg/L), followed by imipenem (4 mg/L), ciprofloxacin (8 mg/L), piperacillin/tazobactam (16 mg/L) and ceftazidime (32 mg/L). These isolates were also analysed genotypically by pulsed-field gel electrophoresis. Five of the seven ICU isolates were identified, one isolate was 98% similar and the other was 85% similar to the ICU isolates. One isolate from the prospective study had approximately 90% genotype similarity to the six ICU isolates with >/=98% similarity. There was no clonality within the strains from the prospective study and clusters with >90% similarity comprised at five or less isolates. Isolates with the same resistance patterns did not necessarily have the same genotypic profile. Strains isolated from different patients on the same day were also not necessarily related. The conclusions of this study were that while the seven ICU isolates were clonal or highly related, they were not widespread throughout Edinburgh and the P. aeruginosa within Edinburgh were highly varied.

Strategies and options for minimizing resistance emergence in pulmonary infections.

The early-onset hospital pulmonary gram-negative infections may respond to ciprofloxacin and co-amoxiclav without significant resistance development. Penicillin-resistant Streptococcus pneumoniae may be treated with macrolides, fluoroquinolones, and glycopeptides. The late-onset hospital pathogens all seem to have developed resistance to cephalosporins, so greater reliance is now made on the fluoroquinolones and carbapenems when aminoglycoside therapy is considered undesirable.

Co-trimoxazole sensitivity testing: comparison of separate and combined disk agar diffusion techniques.

Five hundred and seven strains of bacteria isolated from the urine of patients with significant bacteriuria (more than 10(8) colony-forming units per litre) were tested for sensitivity to co-trimoxazole by the agar diffusion technique. Each organism was tested with a combined disk containing trimethoprim and sulphamethoxazole in a primary sensitivity test and, at a standardised inoculum, with both a combined disk and separate disks of trimethoprim and sulphamethoxazole. The results show that combined disk testing does not always indicate the sensitivity patterns of the organisms being tested.

R-factor mediated trimethoprim resistance: result of two three-month clinical surveys.

All urinary tract isolates were monitored in the Whittington Hospital, London for trimethoprim resistance over a three-month period in 1975; this survey was repeated 18 months later in 1977. In the later survey the incidence of trimethoprim resistance had increased significantly, and the proportion of strains carrying R-factors conferring trimethoprim resistance had nearly doubled. The pattern of resistances associated with R-factor trimethoprim resistance also changed betweeen these two surveys.

TRC-1: emergence of a clavulanic acid-resistant TEM beta-lactamase in a clinical strain.

A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.

SAR-2: identification of a novel plasmid-encoded beta-lactamase from India.

A novel beta-lactamase has been identified in an Escherichia coli strain isolated in South India. The beta-lactamase gene was carried on a plasmid (pUK734) along with resistance determinants to sulphonamides and tetracycline. The novel enzyme has a pI of 8.3 and an Mr of 36,000. The enzyme has a broad-spectrum of activity against both penicillins and cephalosporins. It is also active against oxacillin and methicillin.

TEM-E1: a novel beta-lactamase conferring resistance to ceftazidime.

A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E. coli strain isolated in Belgium. The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7. The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid. However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime.