Mohammad Hamidian

AbaR4 replaces AbaR3 in a carbapenem-resistant Acinetobacter baumannii isolate belonging to global clone 1 from an Australian hospital

OBJECTIVES To explore the diversity of genomic resistance islands in multiply antibiotic-resistant Acinetobacter baumannii isolates in global clone 1 (GC1) from Australian hospitals. METHODS PCR was used to characterize isolates, detect antibiotic resistance genes and insertion sequences and screen for genomic resistance islands. Structures of genomic islands were determined by PCR mapping and sequencing. Multilocus sequence typing was performed using the Oxford scheme. RESULTS Eleven isolates that belong to GC1 were found among 90 A. baumannii isolated between 2001 and 2010 at Australian hospitals, and 5 were carbapenem resistant. Ten isolates had the features characteristic of AbaR3 and related islands, but one carbapenem-resistant isolate did not. Instead, D36 carried the bla(OXA-23) gene in transposon Tn2006, with Tn2006 in AbaR4, and AbaR4 in the chromosomal comM gene, replacing the AbaR3-type island usually associated with multiply antibiotic-resistant GC1 isolates. D36 was resistant to gentamicin, kanamycin and tobramycin due to the aadB gene cassette in the context found in plasmid pRAY and to sulfamethoxazole due to the sul2 gene. D36 was of a rare sequence type (ST), ST247. Bioinformatic analysis identified five potential transposition genes in the AbaR backbone transposons. CONCLUSIONS Substantial diversity was observed among the GC1 isolates. This is the first report of AbaR4 replacing the AbaR3-type island seen in most GC1 isolates, and D36 represents a distinct new GC1 lineage. The AbaRs are members of a large, previously undocumented family of transposons that target comM.

Prevalence and Antimicrobial Resistance of Diarrheagenic Escherichia coli and Shigella Species Associated with Acute Diarrhea in Tehran, Iran

A study was performed to determine the prevalence and antimicrobial resistance of Shigella species and diarrheagenic Escherichia coli isolates cultured from patients with acute diarrhea in Tehran, Iran. Between May 2003 and May 2005, 1120 diarrheal specimens were collected and assayed for bacterial enteropathogens by conventional and molecular methods. Etiological agents were isolated from 564 (50.3%) specimens, and included 305 (54%) E coli, 157 (27.8%) Shigella species, and 102 (18%) from other genera of bacteria. The predominant E coli was Shiga toxin-producing E coli (105 isolates [34.5%]) and the predominant Shigella serotype was Shigella sonnei (88 isolates [56.1%]). A high rate of antibiotic resistance was observed among E coli, with 40 of 53 (75.5%) Shiga toxin-producing E coli isolates resistant to amoxicillin and tetra-cycline, and eight (5.2%) E coli isolates resistant to more than six antibiotics. Most Shigella isolates were resistant to tetracycline (95%) and trimethoprim-sulfamethoxazole (91.7%), with greatest antibiotic resistance observed among S sonnei (53 of 88 [60.2%] isolates). Antibiotic resistance is widespread in diarrheagenic E coli and Shigella in children with acute diarrhea in Tehran, Iran; hence, updated strategies for appropriate use of antimicrobial agents in Iran are needed.

ISAba1 targets a specific position upstream of the intrinsic ampC gene of Acinetobacter baumannii leading to cephalosporin resistance

Sir, Resistance to third-generation cephalosporins such as ceftazidime and cefotaxime in Acinetobacter baumannii is often due to increased expression of an intrinsic ampC gene. Expression increases when the ISAba1 insertion sequence (IS) is present in the appropriate orientation upstream of the ampC gene, providing an outward-facing promoter that appears to be stronger than the intrinsic promoter because it increases transcription. – 3 This mechanism has been found to be widespread, and ISAba1 is generally found upstream of the ampC gene in isolates that are resistant to third-generation cephalosporins. – 8 We have previously shown that most of the isolates in our collection that belong to global clone 1 (GC1) or global clone 2 (GC2), and are resistant to ceftazidime and cefotaxime, carry ISAba1 upstream of ampC. – 7 The precise position of ISAba1 in the isolates examined was determined by sequencing a PCR amplicon that includes the IS and ampC, as described previously. Sequence types were determined using the Oxford multilocus sequence typing scheme as described previously. Initially, two Australian GC2 isolates were examined. The ampC genes of A91, an ST92 isolate from Sydney, 2005 (GenBank accession number JN968483), and RBH44, an ST69 isolate from Brisbane, 2002, differed by a single base substitution, and ISAba1 was in the same orientation and 9 bp away from the ATG initiation codon of ampC in both. ISAba1 is in exactly the same position in the GC1 isolate AYE (GenBank accession number CU459141). However, the insertion events seem to be independent because the ampC alleles, here named allele 1 for GC1 and allele 2 for GC2, differed at 38 or 37 positions in the 1152 bp ampC gene (Table 1). We showed recently that the ampC sequence in GC1 isolates without ISAba1 is identical to that of AYE, which carries ISAba1-ampC. To verify that allele 2 is usually associated with GC2, ampC from A320/RUH134 (isolated in the Netherlands in 1982), which is the oldest GC2 isolate susceptible to third-generation cephalosporins in our collection, was sequenced; the allele (GenBank accession number JN247441) was the same as in the later ISAba1-containing isolates (identical to RBH44 ampC). The shared location of the IS was unexpected, as the promoter identified experimentally by mapping the 5′-end of the transcript is completely contained within ISAba1, and consequently there should be no constraint on the position of the IS to ensure highlevel transcript initiation. The location of this promoter, which includes an extended 210 (TGnCATTAT) that should increase its strength, is shown in Figure S1 (available as Supplementary data at JAC Online). Furthermore, ISAba1 has been shown to transpose to multiple locations. Nonetheless, it appeared that ISAba1 had independently inserted into the same position in both the GC1 and a GC2 background. To further examine the possibility that ISAba1 can target this position, the location of ISAba1 relative to ampC in other sequences available in the GenBank non-redundant DNA database was examined. Two additional cases of ISAba1 associated with distinct ampC alleles were found in accession numbers EU604835 and AY325306. In both of them, the IS was again in the same orientation and separated from the ATG initiation codon of ampC by 9 bp. The number of single nucleotide differences between the various ampC alleles is shown in Table 1.

A GC1 Acinetobacter baumannii isolate carrying AbaR3 and the aminoglycoside resistance transposon TnaphA6 in a conjugative plasmid

Objectives To locate the acquired antibiotic resistance genes, including the amikacin resistance transposon TnaphA6, in the genome of an Australian isolate belonging to Acinetobacter baumannii global clone 1 (GC1). Methods A multiply antibiotic-resistant GC1 isolate harbouring TnaphA6 was sequenced using Illumina HiSeq, and reads were used to generate a de novo assembly and determine multilocus sequence types (STs). PCR was used to assemble the AbaR chromosomal resistance island and a large plasmid carrying TnaphA6. Plasmid DNA sequences were compared with ones available in GenBank. Conjugation experiments were conducted. Results The A. baumannii GC1 isolate G7 was shown to include the AbaR3 antibiotic resistance island. It also contains an 8.7 kb cryptic plasmid, pAb-G7-1, and a 70 100 bp plasmid, pAb-G7-2, carrying TnaphA6. pAb-G7-2 belongs to the Aci6 Acinetobacter plasmid family. It encodes transfer functions and was shown to conjugate. Plasmids related to pAb-G7-2 were detected in further amikacin-resistant GC1 isolates using PCR. From the genome sequence, isolate G7 was ST1 (Institut Pasteur scheme) and ST231 (Oxford scheme). Using Oxford scheme PCR-based methods, the isolate was ST109 and this difference was traced to a single base difference resulting from the inclusion of the original primers in the gpi segment analysed. Conclusions The multiply antibiotic-resistant GC1 isolate G7 carries most of its resistance genes in AbaR3 located in the chromosome. However, TnaphA6 is on a conjugative plasmid, pAb-G7-2. Primers developed to locate TnaphA6 in pAb-G7-2 will simplify the detection of plasmids related to pAb-G7-2 in A. baumannii isolates.

Variants of the gentamicin and tobramycin resistance plasmid pRAY are widely distributed in Acinetobacter

OBJECTIVES To determine the cause of resistance to the aminoglycosides gentamicin and tobramycin in Acinetobacter isolates and the location of the resistance genes. METHODS Australian Acinetobacter baumannii isolates were screened for resistance to aminoglycosides. PCR followed by restriction digestion of amplicons was used to detect genes and plasmids. Plasmids were isolated and examined by restriction digestion. Plasmid DNA sequences were determined and bioinformatic analysis was used to identify features. The sequence of the bla(OXA-Ab) gene and multilocus sequence typing were used to determine strain types. RESULTS Isolates that exhibited resistance to gentamicin, kanamycin and tobramycin were of diverse strain types. These isolates all carried the aadB gene cassette, and in all but one the cassette was in a 6 kb plasmid similar to pRAY. The three plasmid sequences determined revealed multiple frame-shift differences in the available pRAY sequence that altered the reading frames. In pRAY*, mobA and mobC mobilization genes were identified, but no potential replication initiation protein was found. pRAY*-v1 differed from pRAY* by 66 single-base differences, and pRAY*-v2 included two insertion sequences, ISAba22, located upstream of the aadB gene cassette, and IS18-like, within ISAba22. CONCLUSIONS The plasmid pRAY* and variants are widely distributed in Acinetobacter spp. and are the most common cause of resistance to gentamicin and tobramycin. Mobilization genes should assist in the dissemination of pRAY* and its variants.

Five decades of genome evolution in the globally distributed, extensively antibiotic resistant Acinetobacter baumannii global clone 1

The majority of Acinetobacter baumannii isolates that are multiply, extensively and pan-antibiotic resistant belong to two globally disseminated clones, GC1 and GC2, that were first noticed in the 1970s. Here, we investigated microevolution and phylodynamics within GC1 via analysis of 45 whole-genome sequences, including 23 sequenced for this study. The most recent common ancestor of GC1 arose around 1960 and later diverged into two phylogenetically distinct lineages. In the 1970s, the main lineage acquired the AbaR resistance island, conferring resistance to older antibiotics, via a horizontal gene transfer event. We estimate a mutation rate of ∼5 SNPs genome− 1 year− 1 and detected extensive recombination within GC1 genomes, introducing nucleotide diversity into the population at >20 times the substitution rate (the ratio of SNPs introduced by recombination compared with mutation was 22). The recombination events were non-randomly distributed in the genome and created significant diversity within loci encoding outer surface molecules (including the capsular polysaccharide, the outer core lipooligosaccharide and the outer membrane protein CarO), and spread antimicrobial resistance-conferring mutations affecting the gyrA and parC genes and insertion sequence insertions activating the ampC gene. Both GC1 lineages accumulated resistance to newer antibiotics through various genetic mechanisms, including the acquisition of plasmids and transposons or mutations in chromosomal genes. Our data show that GC1 has diversified into multiple successful extensively antibiotic-resistant subclones that differ in their surface structures. This has important implications for all avenues of control, including epidemiological tracking, antimicrobial therapy and vaccination.

Tn6168, a transposon carrying an ISAba1-activated ampC gene and conferring cephalosporin resistance in Acinetobacter baumannii

OBJECTIVES To explore the cause of third-generation cephalosporin resistance in Australian Acinetobacter baumannii isolates belonging to global clone 1 (GC1). METHODS GC1 isolates from Australia were tested for resistance to ceftazidime and cefotaxime using disc diffusion and MICs. PCR was used to determine the context of ISAba1-ampC configurations and amplicons were sequenced. The level of transcripts was measured using quantitative real-time PCR. Multilocus sequence typing was performed. RESULTS All ceftazidime- and cefotaxime-resistant isolates carried an appropriately oriented ISAba1 adjacent to the ampC gene and ISAba1 increased ampC transcripts 8-12-fold. In three isolates, the ampC gene next to ISAba1 was not in the normal chromosomal position. Instead, ISAba1 was 7 bp upstream of an additional copy of ampC located in a 3155 bp duplicated segment of the chromosome that differs from the resident GC1 segment by 2.3% but is almost identical to the corresponding region in several non-GC1 draft genomes. The duplicated segment is bounded by directly oriented copies of ISAba1 and flanked by a 9 bp direct duplication. This 5.5 kb transposon, named Tn6168, is in the same position in the chromosome of the three Australian isolates and the GC1 isolate AB0057. Tn6168 was also detected in an unrelated A. baumannii strain, where it was in a different location. The central part of Tn6168 was probably acquired from a sequence type ST32 (Institut Pasteur scheme) A. baumannii strain. CONCLUSIONS The ISAba1-ampC configuration, which increases ampC expression, can be part of a composite transposon Tn6168.

Horizontal transfer of an ISAba125-activated ampC gene between Acinetobacter baumannii strains leading to cephalosporin resistance

Sir, In Acinetobacter baumannii, resistance to third-generation cephalosporins such as ceftazidime and cefotaxime is known to arise as a consequence of acquisition of an insertion sequence, ISAba1, upstream of the chromosomal ampC gene. Indeed, ISAba1 is frequently found upstream of the ampC gene in isolates that are resistant to third-generation cephalosporins, and the promoter that directs transcription of ampC is located within ISAba1. Other insertion sequences have also been detected upstream of ampC. ISAba125 is present upstream of ampC in ACICU, a global clone 2 (GC2) isolate from Italy, and it has been suggested that it may also increase ampC expression, leading to cephalosporin resistance. We have examined resistance to third-generation cephalosporins in ACICU, kindly supplied by Dr Alessandra Carottoli, Istituto Superiore di Sanita, Rome, Italy, and in A388, a global clone 1 (GC1) isolate from Greece described previously. Both ACICU and A388 were found to be resistant to ceftazidime and cefotaxime (MIC.128 mg/L), but in A388 ISAba1 was not detected upstream of ampC using a PCR assay that links them (Figure 1a). To determine whether a different IS was present in A388, a PCR was designed to detect the ampC gene linked to the folE gene (Figure 1), as it is in its normal location on the chromosome. An amplicon of 1667 bp, the predicted size when no IS is present (Figure 1c), was obtained with GC1 isolates 3208 and D2, which were susceptible to ceftazidime and cefotaxime (MIC 4–16 mg/L). For A388, this amplicon was larger, consistent with the presence of an IS, and the sequence of this folE– ampC amplicon revealed a copy of ISAba125 located 57 bp upstream of the initiation codon of the ampC gene. The amplicons from 3208 and D2 were also sequenced and the sequence of the region surrounding ISAba125 in A388 was identical to those sequences, and to that found in the published genome of the antibiotic-susceptible GC1 isolate AB307-0294 (GenBank accession number CP001172). Because the sequence of ampC found in another GC1 isolate, AB0057 (GenBank accession number CP001182), differed over a short span of 214 bp, the sequence of this region was re-determined and found to be identical to those described here. Hence, it appears that ISAba125 in A388 has inserted into the standard GC1 genome. Examination of the sequence surrounding ISAba125 upstream of the ampC gene in the genome of ACICU (GenBank accession number CP000863), which is from a different clonal complex, namely GC2, revealed that it was almost identical to the GC1 sequence. This suggests that a region derived from a GC1 strain carrying ISAba125 has been incorporated into the ACICU genome. To determine the size of the segment potentially derived from a GC1 strain, the sequence of ACICU in this region was compared with the corresponding region from AB307-0294 and AB0057, representing GC1, and MDR-ZJ06 (GenBank accession number CP001937), representing GC2. A segment of about 9 kb surrounding ISAba125 in ACICU differs from that of other GC2 isolates by about 2%, but is nearly identical to the standard GC1 sequence. Surrounding this segment, the sequences of ACICU and other GC2 strains were the same, but diverged from the GC1 sequence, due to multiple single-nucleotide polymorphisms. On average, GC1 and GC2 sequences differed by 3%. Together these findings indicate that a 9 kb segment of the ACICU genome is likely to have been incorporated into the GC2 lineage from a GC1 strain via homologous recombination. To determine whether the presence of ISAba125 increased the expression of the ampC gene, the RH581–RH582 PCR amplicons from 3208 and from A388 were cloned into pCR2.1TOPO. The MICs of ceftazidime and cefotaxime conferred by the plasmid containing the fragment from 3208 (8 mg/L and 16 mg/L) were 16-fold and 32-fold higher, respectively, than for the vector control (0.5 mg/L). When the cloned fragment contained ISAba125, the MICs were further elevated, to .256 mg/L. To compare the level of ampC transcripts in A388 and ACICU with those in the susceptible isolates, 3208 and D2, quantitative reverse transcription PCR (qRT– PCR) was also performed using primers RH1388, 5′-TGGCTGTGGGT GTTATTCAA-3′, and RH1389, 5′-ACCTGCTGTCGCGGTAAATA-3′, for ampC and RH1384, 5′-GGAGAAAGCAGGGGATCTTC-3′, and RH1385, 5′-ATCCTCTCAGACCCGCTACA-3′, for 16S rRNA. In both A388 and ACICU, the transcript level was 20-fold higher than in 3208 and D2, confirming that the presence of ISAba125 also increased expression of ampC, leading to increased resistance to third-generation cephalosporins. It has previously been shown that incorporation of an ampC gene derived from an A. baumannii strain with an ISOur1 upstream into the genome of Oligella urethralis leads to cephalosporin resistance. Here, we provide evidence for the transfer of cephalosporin resistance from the chromosome of a GC1 isolate to a GC2 strain, raising the possibility that horizontal transfer of resistance determinants that are normally chromosomally located may occur more often than currently envisaged. A 2972 bp DNA sequence from A388 was deposited in GenBank under accession number JQ684178. The sequences of

Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.

IncM Plasmid R1215 Is the Source of Chromosomally Located Regions Containing Multiple Antibiotic Resistance Genes in the Globally Disseminated Acinetobacter baumannii GC1 and GC2 Clones.

Two lineages of extensively antibiotic-resistant A. baumannii currently plaguing modern medicine each acquired resistance to all of the original antibiotics (ampicillin, tetracycline, kanamycin, and sulfonamides) by the end of the 1970s and then became resistant to antibiotics from newer families after they were introduced in the 1980s. Here, we show that, in both of the dominant globally disseminated A. baumannii clones, a related set of antibiotic resistance genes was acquired together from the same resistance region that had already evolved in an IncM plasmid. In both cases, the action of IS26 was important in this process, but homologous recombination was also involved. The findings highlight the fact that complex regions carrying several resistance genes can evolve in one location or organism and all or part of the evolved region can then move to other locations and other organisms, conferring resistance to several antibiotics in a single step. ABSTRACT Clear similarities between antibiotic resistance islands in the chromosomes of extensively antibiotic-resistant isolates from the two dominant, globally distributed Acinetobacter baumannii clones, GC1 and GC2, suggest a common origin. A close relative of the likely progenitor of both of these regions was found in R1215, a conjugative IncM plasmid from a Serratia marcescens strain isolated prior to 1980. The 37.8-kb resistance region in R1215 lies within the mucB gene and includes aacC1, aadA1, aphA1b, blaTEM, catA1, sul1, and tetA(A), genes that confer resistance to gentamicin, streptomycin and spectinomycin, kanamycin and neomycin, ampicillin, chloramphenicol, sulfamethoxazole, and tetracycline, respectively. The backbone of this region is derived from Tn1721 and is interrupted by a hybrid Tn2670 (Tn21)-Tn1696-type transposon, Tn6020, and an incomplete Tn1. After minor rearrangements, this R1215 resistance island can generate AbGRI2-0*, the predicted earliest form of the IS26-bounded AbGRI2-type resistance island of GC2 isolates, and to the multiple antibiotic resistance region (MARR) of AbaR0, the precursor of this region in AbaR-type resistance islands in the GC1 group. A 29.9-kb circle excised by IS26 has been inserted into the A. baumannii chromosome to generate AbGRI2-0*. To create the MARR of AbaR0, a different circular form, again generated by IS26 from an R1215 resistance region variant, has been opened at a different point by recombination with a copy of the sul1 gene already present in the AbaR precursor. Recent IncM plasmids related to R1215 have a variant resistance island containing a blaSHV gene in the same location. IMPORTANCE Two lineages of extensively antibiotic-resistant A. baumannii currently plaguing modern medicine each acquired resistance to all of the original antibiotics (ampicillin, tetracycline, kanamycin, and sulfonamides) by the end of the 1970s and then became resistant to antibiotics from newer families after they were introduced in the 1980s. Here, we show that, in both of the dominant globally disseminated A. baumannii clones, a related set of antibiotic resistance genes was acquired together from the same resistance region that had already evolved in an IncM plasmid. In both cases, the action of IS26 was important in this process, but homologous recombination was also involved. The findings highlight the fact that complex regions carrying several resistance genes can evolve in one location or organism and all or part of the evolved region can then move to other locations and other organisms, conferring resistance to several antibiotics in a single step.