Emergence of blaKPC carbapenemase genes in Australia.

blaKPC genes encoding resistance to carbapenems are increasingly widely reported and are now endemic in parts of several countries, but only one Klebsiella pneumoniae isolate carrying blaKPC-2 had previously been reported in Australia, in 2010. Here we characterised this isolate, six additional K. pneumoniae and one Escherichia coli carrying blaKPC and another K. pneumoniae lacking blaKPC, all isolated in Australia in 2012. Seven K. pneumoniae belonged to clonal complex (CC) 292, associated with blaKPC in several countries. Five with blaKPC-2 plus the isolate lacking a blaKPC gene were sequence type 258 (ST258) and the seventh was the closely related ST512 with blaKPC-3. The eighth K. pneumoniae isolate, novel ST1048, and the E. coli (ST131) also carried blaKPC-2. blaKPC genes were associated with the most common Tn4401a variant, which gives the highest levels of expression, in all isolates. The ST258 isolates appeared to share a similar set of plasmids, with IncFIIK, IncX3 and ColE-type plasmids identified in most isolates. All K. pneumoniae isolates had a characteristic insertion in the ompK35 gene resulting in a frameshift and early termination, but only the ST512 isolate had a GlyAsp insertion in loop 3 of OmpK36 that may contribute to increased resistance. The clinical epidemiology of blaKPC emergence in Australia thus appears to reflect the global dominance of K. pneumoniae CC292 (and perhaps E. coli ST131). Some, but not all, patients carrying these isolates had previously been hospitalised outside Australia, suggesting multiple discrete importation events of closely related strains, as well as undetected nosocomial spread.

Simple multiplex real-time PCR for rapid detection of common 16S rRNA methyltransferase genes.

We have developed a real-time multiplex PCR assay to detect the three most common 16S rRNA methyltransferase genes (armA, rmtB and rmtC), which encode problematic high-level resistance to all clinically-relevant aminoglycoside antibiotics. All results were consistent with published conventional PCR assays and these genes still appear rare in Australia.

Prediction of major antibiotic resistance in Escherichia coli and Klebsiella pneumoniae in Singapore, USA and China using a limited set of gene targets.

Antibiotic resistance in Gram-negative bacteria, especially Enterobacteriaceae, can be conferred by a large number of different acquired resistance genes, although it appears that relatively few dominate. A previous survey of Escherichia coli and Klebsiella pneumoniae isolates from Sydney, Australia, revealed that a limited set of genes could reliably predict resistance to third-generation cephalosporins (3GCs) and aminoglycosides. Here we tested E. coli and K. pneumoniae isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration of ≥ 2 µg/mL from China and Singapore, with significantly higher resistance rates than Australia, as well as the USA. Few targets were needed to predict non-susceptibility to 3GCs (95/95; 100%) and gentamicin (47/51; 92%). The gene types detected here are consistent with previous surveys in similar countries with similar resistance rates, where the majority of 3GC resistance can be explained by blaCTX-M genes. This study identified a limited set of genes capable of predicting resistance to 3GC and aminoglycoside antibiotics and implies a restriction in the global resistance gene pool that can be exploited for diagnostic purposes.

Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae.

Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥ 2 µg/mL were collected from seven major hospitals in Sydney (Australia) in 2008-2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC ≥ 2 µg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC ≥ 8 µg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5-10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.

Genetic diversity and antibiotic resistance in Escherichia coli from environmental surface water in Dhaka City, Bangladesh.

The extended-spectrum ß-lactamase gene bla(CTX-M-15) was almost ubiquitous in diverse antibiotic-resistant Escherichia coli isolated from surface water around Dhaka City, Bangladesh. Forty-eight isolates represented 34 multi-locus sequence types and a variety of plasmid replicons were identified in association with bla(CTX-M-15) and other resistance genes. This water is likely to be an important source of transmissible antibiotic resistance in Bangladesh.

The ecology of antibiotic use in the ICU: homogeneous prescribing of cefepime but not tazocin selects for antibiotic resistant infection.

BACKGROUND: Antibiotic homogeneity is thought to drive resistance but in vivo data are lacking. In this study, we determined the impact of antibiotic homogeneity per se, and of cefepime versus antipseudomonal penicillin/ß-lactamase inhibitor combinations (APP-ß), on the likelihood of infection or colonisation with antibiotic resistant bacteria and/or two commonly resistant nosocomial pathogens (methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa). A secondary question was whether antibiotic cycling was associated with adverse outcomes including mortality, length of stay, and antibiotic resistance. METHODS: We evaluated clinical and microbiological outcomes in two similar metropolitan ICUs, which both alternated cefepime with APP-ß in four-month cycles. All microbiological isolates and commensal samples were analysed for the presence of antibiotic-resistant bacteria including MRSA and P. aeruginosa. RESULTS: Length of stay, mortality and overall antibiotic resistance were unchanged after sixteen months. However, increased colonisation and infection by antibiotic-resistant bacteria were observed in cefepime cycles, returning to baseline in APP-ß cycles. Cefepime was the strongest risk factor for acquisition of antibiotic-resistant infection. CONCLUSIONS: Ecological effects of different ß-lactam antibiotics may be more important than specific activity against the causative agents or the effect of antibiotic homogeneity in selection for antibiotic resistance. This has important implications for antibiotic policy.

A novel gene cassette, aacA43, in a plasmid-borne class 1 integron.

A novel gene cassette, aacA43, was identified in the aadB-aacA43-oxa10-smr2 cassette array in a class 1 integron. Like related aminoglycoside-(6')-acetyltransferases, AacA43 confers clinically relevant resistance to kanamycin, tobramycin, and some less-used aminoglycosides but not to gentamicin. Although transferable on an IncL/M plasmid, aacA43 was identified in only two different Klebsiella pneumoniae strains (14 isolates), one Escherichia coli strain (2 isolates), and one Enterobacter cloacae strain in a survey of patients in a Sydney intensive care unit in 2004-2005.

Quantitative analysis of gene-specific DNA damage in human spermatozoa.

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H2O2; 0-5 mM) or iron (as Fe(II)SO4, 0-500 microM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, beta-pol and beta-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H2O2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H2O2. The mitochondrial genome of human spermatozoa was significantly (P<0.001) more susceptible to H2O2-induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage.