The above image is taken from a Gram stained smear prepared from a blood culture bottle. It shows what appears to be Gram positive cocci in chains. The next day, grey nondescript colonies were seen on the blood agar plates which could have passed for Streptococcus spp. The staff were therefore surprised when the MALDI-TOF identified the organism as Moraxella spp.
A Gram stain made from colonies on the blood agar plates showed fat Gram negative cocco-bacilli typical of Moraxella spp (above).
Of interest, if you make a Gram stain smear from colonies growing around a penicillin disk (above), Moraxella spp. will form long filaments or spindle-shaped cells(below). This is known as the penicillin disk test (Caitlin BW, 1975), which can be used to quickly differentiate between Moraxella spp. and Neisseria spp (which are also Gram negative cocci but do not change their Gram stain appearance when under the influence of sub-inhibitory concentrations of penicillin).
When a repeat Gram-stained smear was made from the blood culture bottle with special attention paid to the decolorization step with acetone-alcohol, there were fewer Gram positive cells seen (below). Moraxella spp. sometimes resist decolorization and may confuse microbiologists by appearing Gram positive!
First, blaIMI-1 was one of the earliest carbepenemase genes to be described (even before carbapenems were in widespread use).
Second, it seems to be only associated with the Enterobacter cloacae complex (ECC) and not any other bacteria. At the same time not every ECC carries blaIMI-1 so it is not a gene normally found in this species.
How do ECC acquire this gene then? Previous studies have shown that blaIMI-1 was not on a plasmid so it must be on the chromosome. Did it get there by a transposon or an integron mechanism? But these gene mobilization methods are quite generalized and would not explain the specific association between blaIMI-1 and ECC. Furthermore no one appeared to have sequenced the flanking regions of blaIMI-1 to show its genetic environment, which could give a clue as to how it was mobilized.
It was only with the availability of whole genome sequencing that I started entertaining the idea of investigating this. (This was also prompted by an increase in the number of blaIMI-1 positive ECC being isolated in our lab but that is the subject of a future post).
That was in 2013. We didn’t actually send off the initial batch of isolates for WGS till circa Oct 2016! (At the time blaIMI-1 was still vanishingly rare worldwide and we didn’t think there was any competition-so no rush). When we got the sequences back we initially didn’t recognize any of the usual mobile elements. While doing a BLAST search of the sequences against the GenBank database we noticed a close match with the database entry (GenBank accession number KU870982.1)
of an
ECC sequence which had been submitted in March 2016 (though the Canadian authors had omitted any mention of blaIMI-1 in the title). So there was competition after all!
It was our collaborator Dr J Teo at the National University Hospital who pointed out that blaIMI-1 appeared to be associated with an Integrative Mobile Element that exploited the Xer machinery (IMEX).
She also found an article describing IMEX elements with blaNMC-A (NMC-A is a carbapenemase very closely related to IMI-1) inserted into the chromosome of members of the ECC (Antonelli A, 2015).
I of course had never heard of IMEX before and was therefore obliged to do a literature search. This fortunately was quite short because the role of IMEX-elements in acquired antimicrobial resistance is relatively recently described.
Because bacterial chromosomes are circular, the replication of DNA during cell division may result in the sister chromosomes being interlinked as in a chain (catenanes), or joined end to end (dimers) (See Midonet C, 2014 for illustrative figures).
Catenanes are resolved by Topoisomerase IV. A separate mechanism, the Xer machinery can resolve both catenanes and dimers.
Most
bacteria
encode the tyrosine recombinases XerC and XerD in their genomes. XerC and XerD resolve dimers by addition of a crossover at specific sites in the genome (known as dif sites). The
blaIMI-1 gene is on a mobile element that has hijacked this mechanism to integrate into the genome ofECC at these dif sites. This represents a form of site-specific recombination and presumably the site-specificity and dependence on the Xer machinery explain why so far blaIMI-1 is only found in ECC (and even then only rarely).
While we were still analyzing our sequences, the Canadians went to press (Boyd DA, 2017).
From 2010 to 2015, the National Microbiology Laboratory, Public Health Agency of Canada received 19 blaNMC-A or blaIMI-type positive ECC isolates. The carbapenemase genes of all NMC-A (n=10), IMI-1 (n=5), and IMI-9 (n=2) producers were found in IMEX elements.Two novel genes, blaIMI-5 and blaIMI-6, were found on plasmids.
In our own hospital we isolated 16 blaIMI-1 positive ECC between April 2013 and March 2015.The blaIMI-type genes were all found on IMEX. The IMEX of 5 isolates were similar to those described in Canada, while the remainder were novel (Koh TH, 2017).
So we have added supportive data to previous studies associating the mobilization of blaNMC-A and blaIMI-type genes into the ECC with IMEX elements. This still begs the question where these genes originate from. Together with the results of the Canadian study, the geographical distribution, diversity of the ECC isolates, and the heterogeneity of their IMEX elements hint at a potentially widely distributed environmental source.
Das B, Martínez E, Midonet C, Barre FX. Integrative mobile elements exploiting Xer recombination. Trends Microbiol. 2013 Jan;21(1):23-30. (no free access)
Koh TH, Rahman NBA, Teo JWP, La MV, Periaswamy B, Chen SL. Putative Integrative Mobile Elements That Exploit the Xer Recombination Machinery Carrying blaIMI-Type Carbapenemase Genes in Enterobacter cloacae Complex Isolates in Singapore. Antimicrob Agents Chemother. 2017 Dec 21;62(1). pii: e01542-17. (no free access until Jan 2018)
The carbapenemase genes in the preceding 2 posts (blaNDM-1 and blaOXA-48-like) were a bit of a ‘surprise bonus’, in the sense that we had not been expecting them when they appeared.
Within a decade, KPC-producing Enterobacteriaceaerapidly spread to other parts of the United States (Kitchel B, 2009). Eventually they also started to emerge elsewhere particularly Israel, China, and some countries in Europe and South America (Nordmann P, 2009).
The first KPC-2 producers in Singapore were isolated in June and July 2011 and were described by colleagues at the National University Hospital (Balm M, 2012). All patients had no recent travel history. So unlike the case of blaNDM-1, and blaOXA-181, blaKPC-2 seems to have quietly sneaked in (like blaOXA-48), and we are not able to determine the exact time point that it was first introduced into Singapore. Interestingly, the MLST sequence type of the K. pneumoniae isolates and the genetic environment of the blaKPC-2 gene suggested a source from China rather the USA or Europe.
We isolated the first KPC-producing K. pneumoniae in our own hospital in Dec 2011 (missed a hattrick by 6 months!).
Initially at least, many of our KPC-producing Enterobacteriaceae were less resistant to the other non-beta-lactam antibiotics like amikacin (AN) and gentamicin (GM) compared to those producing NDM-1 or OXA-181 carbapenemases.
The modified Hodge test is obviously positive for KPC- producers. The isolate under test is arrowed, but in fact our positive control (streak at bottom right) is also a KPC-producer.
KPC is inhibited by boronic acid hence there is enhancement of the zone diameter around the disc containing meropenem and boronic acid (MR+BO).
Both the Carba NP (above) and Rapid Carb Blue (below) readily detect carbapenemase production (color change from red to yellow) in blaKPC-positive Enterobacteriaceae.
Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis. 2009 Apr;9(4):228-36. (no free access)
In September 2011, the year following the introduction of blaNDM-1 to Singapore we isolated another multidrug-resistant Klebsiella pneumoniae which was also resistant to all antibiotics tested.
Within 2 weeks we were referred another K. pneumoniae, with the same antibiotic susceptibility profile, from a patient admitted to another hospital. Both patients had been in the same hospital in Bangladesh before coming to Singapore to seek medical attention.
In fact we were able to show by molecular fingerprinting (using pulsed-field gel electrophoresis) that both isolates were identical and therefore probably part of an outbreak clone in that particular hospital in Bangladesh .
While these isolates looked as resistant as the ones producing NDM-1, they were negative for the blaNDM-1 by PCR. They had blaOXA-181 which was a new carbapenemase gene that had only been described 7 months earlier (Castanheira M, 2011).
Unlike NDM-1, OXA-181 displays obvious carbapenemase activity with the Modified Hodge Test.
None of the chemicals in the ROSCO test enhance the activity of meropenem against an OXA-181 producer.
OXA-181 belongs to the OXA-48-like family of Ambler Class D carbapenemases. OXA-48 producing K. pneumoniae first emerged as a problem in Turkey in the early 2000s (Poirel L, 2004), but have since spread to North Africa, Europe and the rest of the world (Poirel L, 2012).
In our experience, the true OXA-48 producers tend to be a bit harder to detect in our lab compared to producers of the OXA-48-like carbapenemases like OXA-181 and OXA-232 (Teo JW, 2013). This is because the latter tend to produce other beta-lactamases and are more obviously multi-drug resistant.
We isolated our first true OXA-48 producer (an Escherichia coli) in Jan 2012. The 2 pictures below actually show our second isolate which was a K. pneumoniae isolated in May 2012. Both were isolated from local Chinese patients with no apparent travel history.
What is striking about this OXA-48 producing K. pneumoniae is that there are still zones of inhibition around the imipenem (IPM) and ertapenem (ETP) discs. In addition there are large zones of inhibition around the ceftriaxone (CRO) and aztreonam (ATM) discs. OXA-48 spares third generation cephalosporins.
The carbapenemase activity of OXA-48 in this isolate (arrow) is still obvious by the modified Hodge test.
In our hands the carba NP sometimes has some problems detecting OXA-48-like producers. (A color change from red to yellow is supposed to indicate the presence of a carbapenemase.)
We get better results with the Rapid Carb Blue. (A change in color from green-blue to yellow indicates the isolate that has been inoculated produces a carbapenemase, negative controls are the left of each pair of tubes.)
In a previous post, I hinted that the plasmid-borne carbapenemase gene blaIMP-1 did not signal the end of the antibiotic era as feared and that blaIMP-1 would eventually be eclipsed by the emergence of newer carbapenemase genes.
The first of these arrived in our laboratory in January 2010 (during the intervening 14 years, the laboratory only detected one other
blaIMP-1-positive Enterobacteriaceae).
This was the most resistant Enterobacteriaceae I had ever encountered (see below)!
Essentially this Klebsiella pneumoniae was resistant to every antibiotic we tested including the carbapenems.
The imipenem (IP) MIC decreased from 32 mg/L to < 1 mg/L in the presence of the metal chelator EDTA (IPI) indicating that the carbapenemase involved was a metallo-beta-lactamase.
The following month, we isolated a second K. pneumoniae with a similar antibiogram.
Surprisingly, the multiplex PCR we were using that targeted the main metallo-beta-lactamase genes known at the time (primarily blaIMP and blaVIM) was negative for both isolates (Ellington MJ, 2007).
Nowadays we would just send this isolate for whole-genome sequencing but at that time this was not a viable option for us. So the cause of this multidrug resistant phenotype continued to remain a mystery until the end of June 2010.
I had not heard of NDM-1 before but I could sense a light bulb flashing in my head. According to the MMWR paper, NDM-1 producing bacteria in the UK and USA were associated with patients with a travel history to the Indian subcontinent (CDC, 2010). A quick check on our own 2 patients showed that the first had received medical attention in India, and the second was from Bangladesh.
I quickly dug up the original NDM-1 paper which had in fact only been published a month before our first isolate (Yong D, 2009). We ordered a set of blaNDM-1 PCR primers and ran them on DNA extracts from our 2 isolates—nothing, no PCR product. Fortunately, a change of PCR mastermix sorted this out and we were able to confirm the presence of blaNDM-1 in both isolates by sequencing the PCR products (Koh TH, 2010). This was just as well as the Lancet Infectious Diseases had just published a paper on the spread of blaNDM-1 from the Indian subcontinent to the UK (KumarasamyKK, 2010). This had caught the attention of the mainstream media and even government health agencies were now interested in NDM-1 (Straits Times, 2010).
blaNDM-1 can happily coexist with several other antibiotic resistance genes. In each of our first 2 isolates, it was found together with
1 ESBL (blaCTX-M Group 1) and 2 plasmid ampC (blaCMY and blaDHA) genes!
In some ways blaNDM-1 is quite remarkable. It is often found on plasmids that are very promiscuous and therefore disseminates very well to other bacteria in the environment (Walsh TR, 2011).
Defining an outbreak of NDM-1 producing bacteria is difficult as spread can be at the plasmid level as well as the bacterial strain level. Investigation of such outbreaks often requires newer typing techniques that involve next generation sequencing (Khong WX, 2016).
Modified Hodge test (MHT) for detection of carbapenemase production. A meropenem disc (MEM 10) is placed on a lawn of susceptible Escherichia coli. Test and control bacterial isolates are streaked out from the meropenem disc and the plate is incubated overnight. The meropenem diffuses out from the disc into the agar resulting in a zone of inhibition. Carbapenemase production by the positive control (bottom streak) allows the E. coli to grow into the zone of inhibition along the positive control streak resulting in a ‘clover-leaf’ appearance. This contrasts with the negative control (top streak) where there is no ingrowth along the streak. The NDM-producing test strain is on the left (arrow). NDM-1 producers are notorious for giving very weak (or even negative) MHT results.
ROSCO discs. MRP10 (meropenem), MRPCX (meropenem+cloxacillin), MRPBO (meropenem+boronic acid), MRPDP (meropenem+dipicolinic acid). Enhancement of the zone of inhibition around the MRPDP disc shows that NDM-1 is a metallo-beta-lactamase. Dipicolinic acid (like EDTA) is chelating the zinc ions required for metallo-beta-lactamase activity. (The temocillin disc has been retouched out of the photo).
The combination of a weak or negative MHT result with enhancement of the zone of inhibition around MRPDP is highly suggestive of an NDM-1 producer.
Because of technical issues interpreting the MHT, a number of alternative methods for detecting carbapenemase production have been developed. In the Carba NP, a color change from red to yellow indicates the presence of a carbapenemase (Nordmann P, 2012).
The Rapid Carb Blue is done in pairs with one tube containing a negative control (without carbapenem). A change in color from green-blue to yellow in the other tube (containing carbapenem) indicates the isolate that has been inoculated into the tube produces a carbapenemase.
Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis. 2011 May;11(5):355-62. (no free access)
I first heard about Staphylococcus argenteus when we were investigating an outbreak of MRSA in laboratory monkeys (see previous post).
In the initial stages of the outbreak, the author of MIPHIDIC opined that we could be dealing with the newly described Staphylococcus
schweitzeri, which had been isolated from a dead Black-cheeked White-nosed Monkey (Cercopithecus ascanius) from Gabon (Tong SYC, 2015), or Staphylococcus argenteus which had been isolated from west lowland Gorillas (Schuster D, 2016).
Although this turned out not to be be the case, I was interested to learn that these 2 newly described species of coagulase-positive Staphylococcus spp. were phenotypically difficult to distinguish from Staphylococcus aureus but were highly divergent (~10%) at the genome level (though the 16S ribosomal RNA gene sequences are similar).
S. argenteus is non-pigmented, lacking the genes to produce the carotenoid pigment staphyloxanthin which gives S. aureus colonies their characteristic ‘golden’ appearance (Holt DC, 2011).
Since staphyloxanthin is considered a pathogenicity factor, it was originally thought that S. argenteus was relatively harmless. However it probably causes infections similar to S. aureus (Thaipadungpanit J, 2015). A study found that S. argenteus was an important cause of community-acquired invasive sepsis in Thailand, where it seemed to cause less respiratory failure compared to S. aureus sepsis (Chantratita N, 2016). Recently it has been blamed as the cause of a food poisoning incident though the isolates recovered contained accessory genes not typically found in previous S. argenteus isolates (Suzuki Y, 2017).
I was quite keen to get hold of a S. argenteus isolate and was excited to come across an article which described 2 isolates from Singapore (Moradigaravand D, 2017). Unfortunately on contacting my colleague whose laboratory had contributed those isolates, I was disappointed to learn that he had not archived them.
Fortuitously, another hospital in Singapore has been sending us S. aureus isolates for study. I was delighted to find that one of these isolates had the multilocus sequence type ST2250 which is the most common type of S. argenteus found in Thailand
(Moradigaravand D, 2017).
This was isolated from a baby born to local parents. Both MALDI-TOF and Vitek 2 identified the isolate as S. aureus.
The difference in the color of the colonies of S. argenteus (left) and S, aureus (right) when grown on Trypticase soy agar with 5% added sheep blood is not obvious.
The difference in pigmentation between S. argenteus (left) and S, aureus (right) is much more obvious on chocolate agar.
Close-up of S. argenteus on Trypticase soy agar with 5% added sheep blood viewed with reflected light.
Close-up of S. argenteus on Trypticase soy agar with 5% added sheep blood viewed with transmitted light showing beta-haemolysis.
S. argenteus is positive for latex agglutination like S. aureus.
Gram stain of S. argenteus.
While distinct species, S. argenteus and S. aureus are able to exchange virulence and antimicrobial resistance genes. Therefore based on present knowledge, while interesting from an evolutionary and epidemiological perspective, I’m not sure if it is clinically important to separate S. argenteus from S. aureus in routine lab practice.
Schuster D, Rickmeyer J, Gajdiss M, Thye T, Lorenzen S, Reif M, Josten M, Szekat C, Melo LD, Schmithausen RM, Liégeois F, Sahl HG, Gonzalez JJ, Nagel M, Bierbaum G. Differentiation of Staphylococcus argenteus (formerly: Staphylococcus aureus clonal complex 75) by mass spectrometry from S. aureus using the first strain isolated from a wild African great ape. Int J Med Microbiol. 2017 Jan;307(1):57-63. (no free access)
Suzuki Y, Kubota H, Ono HK, Kobayashi M, Murauchi K, Kato R, Hirai A, Sadamasu K. Food poisoning outbreak in Tokyo, Japan caused by Staphylococcus argenteus. Int J Food Microbiol. 2017 Dec 4;262:31-37. (no free access)
A middle aged man presented with fever and signs of meningism. He worked in the roasted meat section of a Chinese restaurant.
Blood cultures were positive.
Fig 1. Gram stain of slide prepared from aerobic blood culture bottle.
Fig 2. Gram stain of slide prepared from anaerobic blood culture bottle.
Fig 3. Growth on trypticase soy agar with 5% sheep blood.
Fig 4. Close up of alpha-haemolytic colonies viewed with reflected light.
Fig. Close up of colonies viewed with transmitted light.
The white cell count in the cerebrospinal fluid (CSF) was elevated at 57/microliter. The CSF/Blood glucose ratio was low at 0.25. The total protein was elevated at >2.0 g/L. The same organism was grown from the cerebrospinal fluid culture.
The isolates were identified as Streptococcus suis by MALDI-TOF.
S. suis is an important pathogen of pigs, causing pneumonia, meningitis, septicaemia, and septic arthritis. It can also colonize pigs asymptomatically in the upper respiratory (particularly the tonsils) and gastrointestinal tracts.
Zoonotic infections happen to people that handle pigs or pork e.g. pig farmers, abattoir workers, cooks. In humans, S. suis usually causes meningitis, and septicaemia. The ear is often affected causing hearing loss, loss of coordination, and dizziness.
The largest recorded outbreak of S. suis infection in humans occurred in China in 2005 (Yu H, 2006). There were 215 cases recorded and 39 deaths.The cause was traced to the local farming practice of slaughtering sick pigs for human consumption.
S. suis is also a common cause of community meningitis and bacteraemia in Vietnam where it has been associated with the consumption of raw pork products (Huong VT, 2014).
Because Singapore no longer has any pig farms, S. suis is only isolated sporadically.
If you read some of the references from our previous post, you will have realized that besides ESBL production, there is another mechanism by which Enterobacteriaceae may develop oxyimino-cephalosporin (e.g. ceftriaxone and ceftazidime) resistance in Singapore (Koh TH, 2004 and 2008).
The AmpC beta-lactamases are cephalosporinases that are actually naturally found in almost all important Enterobacteriaceae with the notable exception of Klebsiella and Salmonella spp. All Escherichia coli for example have an ampC gene in their chromosome but it is usually not expressed so it is usually considered an insignificant contributor to antimicrobial resistance.
There is however a group of Enterobacteriaceae (known as
the ESCHAPPM Group) where the chromosomal ampC gene may make a significant contribution to antimicrobial resistance. ESCHAPPM is a mnemonic for
Enterobacter cloacae complex, Enterobacter aerogenes, Serratia marcescens,
Citrobacter freundii complex, Hafnia alvei, Aeromonas hydrophila, Aeromonas
caviae, Aeromonas veronii, Providencia stuartii, Providencia rettgeri and Morganella morganii (the most clinically significant species are in bold).
Fig 1. A member of the Enterobacter cloacae complex. Why are the zones of inhibition around the ceftriaxone (CRO) and aztreonam (ATM) discs distorted? See explanation below.
In all these bacteria, AmpC production is inducible i.e. some antibiotics can cause production of the beta-lactamase to increase. So for example in Fig 1 above, amoxicillin-clavulanate (AMC), imipenem (IPM), and ertapenem (ETP) are inducing production of AmpC in the bacteria between them and the CRO and ATM discs. AmpC hydrolyzes CRO and ATM. This leads to the zones of inhibition becoming smaller, or truncated (4 black lines), around the CRO and ATM discs opposite IPM, AMC, and ETP.
AMC itself is hydrolyzed by AmpC and therefore there is no zone of inhibition around the AMC disc. IPM and ETP are not hydrolyzed by AmpC and therefore the zones of inhibition are not reduced.
Can you use ceftriaxone (or aztreonam) to treat a patient infected with this isolate? Ceftriaxone by itself does not induce production of AmpC so it would appear to be safe. However it is not the inducibility of the AmpC that is the issue.
Notice the 3 tiny colonies (black arrows) in the zone of inhibition around the CRO disc? These are derepressed mutants that hyper-produce AmpC (i.e. the regulatory mechanism for controlling the amount of AmpC enzyme has been disrupted. AmpC production does not need to be induced and these bacteria are producing large amounts of AmpC all the time).
The key factor is how frequently these derepressed mutants are likely to occur. This differs according to the species of bacteria and therefore correct laboratory identification has clinical implications. A nice summary can be found on the Australian CDS website.
The Enterobacter cloacae complex, Enterobacter aerogenes, and the Citrobacter freundii complex have a high frequency (10-5 to 10-6) of developing mutants.
S. marcescens, does not give rise to derepressed mutants when exposed to ceftazidime, piperacillin-tazobactam and aztreonam but does so when exposed to other β-lactams.
Aeromonas spp. (with the exception of A. sobrii and A. veronii which do not produce this enzyme) produce derepressed mutants at variable frequencies. What is interesting is that ceftazidime and aztreonam do not seem to trigger mutant formation. Even when derepressed mutants are produced by exposure to other antibiotics, they remain susceptible to ceftazidime and aztreonam.
In H. alvei, P. stuartii, P. rettgeri and M. morganii,
derepressed mutants hyper-producing AmpC are only selected at a very low mutation rate of
10-8
.
In some cases, inducible AmpC beta-lactamase genes have escaped from their original hosts onto plasmids (pAmpC) and been acquired by other bacteria.
Fig 2.
The above is a K. pneumoniae positive for the pAmpC gene blaDHA.
It behaves exactly like E. cloacae complex with a chromosomal AmpC and illustrates the risk of antibiotic failure posed by the development of derepressed mutants.
Fig 3. There are many colonies of derepressed mutants around the ceftazidime (CAZ) disc.
Fig 4. Here we have done an ceftriaxone (TX) Etest with a derepressed mutant on the left of the agar plate and the original isolate on the right. You can see that the original isolate has a ceftriaxone minimal inhibitory concentration of about 1 mcg/ml (susceptible) whereas the derepressed mutant is >32 mcg/ml (resistant).
As you can see, it is quite easy for labs that are unaware of the nuances of inducible AmpC producing bacteria to report them as susceptible to oxyimino-cephalosporins.
In a situation where the patient is very unwell, or there is likely to be a high inoculum of bacteria, clinicians should be advised not to use oxyimino-cephalosporins (like ceftriaxone) to treat infections with bacteria that have a high frequency of developing derepressed mutants hyper-producing AmpC enzymes.
Fig 5. AmpC and pAmpC are weak carbapenemases and can confer resistance to carbapenems in combination with diminished porin expression. In fact before Enterobacteriaciae producing the true carbapenemases like NDM-1, KPC-2 and OXA-48-like became more common in Singapore, most carbapenem resistant Enterobacteriaceae isolated in our lab were
blaDHA-positive K. pneumoniae (above).
Besides blaDHA
in K. pneumoniae, there is another major acquired pAmpC in Singapore (Koh TH, 2007).
This is blaCMY, most often found in E. coli (this is distinct from the chromosomal AmpC gene that is intrinsic to E. coli but is usually not expressed). CMY-producing E. coli are less of a therapeutic problem compared with DHA-producing K. pneumoniae because this pAmpC is constitutively produced and the bacteria are obviously resistant (or intermediately resistant) to oxyimino-cephalosporins. Consequently, because the lab has no problem identifying antibiotic resistance, the patient is unlikely to receive inappropriate antibiotics.
The main issue with CMY-producing E. coli is that they are associated with farm animals and represent a supply of oxyimino-cephalosporin resistant Enterobacteriaceae that can circulate in the community (Winkur, 2001).
Koh TH, Sng LH, Wang G, Hsu LY, Lin RT, Tee NW. Emerging problems with plasmid-mediated DHA and CMY AmpC beta-lactamases in Enterobacteriaceae in Singapore. Int J Antimicrob Agents. 2007 Sep;30(3):278-80. (no free access)
Figure 1. Key-hole effect showing augmentation of activity of ceftriaxone and aztreonam by clavulanic acid diffusing from the amoxicillin-clavulanate disc.This demonstrates that this E. coli from chicken meat being tested produces an ESBL. AM: ampicillin; CF: cephalothin; CRO: ceftriaxone; AMC: amoxicillin-clavulanic acid; ATM: aztreonam; MEM: meropenem.
Much of
the attention surrounding antibiotic resistance in Gram-negative bacilli is now
focused on carbapenemase-producing Enterobacteriaceae (CPE) (see a previous post). However the
problem with CPE has partly arisen because of the use of carbapenem
antiibiotics to treat infections with a previous generation of multi-drug
resistant Enterobacteriaceae resistant to oxyimino-cephalosporins (e.g. ceftriaxone, cefotaxime, ceftazidime) -
principally those that produce extended-spectrum beta-lactamases (ESBL).
These
started to emerge in Singapore around the late 1980s-early 1990s (Koh TH, 2008).
Though extensive molecular studies were not carried out on those early
isolates, they were phenotypically suggestive of TEM and SHV ESBLs, typically
being more resistant to ceftazidime than ceftriaxone.
In the
late 1990s, local microbiologists started to notice a change with increasing
numbers of ESBL-producing Enterobacteriaceae being isolated that were resistant
to ceftriaxone but apparently
susceptible to ceftazidime. These turned out to be producing
a different type of ESBL known as CTX-M beta-lactamases, and have in
fact since displaced the old TEM and SHV-producers (Koh TH, 2004).
This was
not just an academic observation without practical implications. Whereas the
earlier TEM and SHV type ESBL-producing Enterobacteriaceae were largely
confined to hospitalized patients, some of these newer ESBL-producing
Enterobacteriaceae seemed to be coming in with patients from the community with
no previous healthcare contact (Young BE, 2014). Where could these patients be
getting these resistant bacteria from?
In fact
for some time, microbiologists in other countries have reported CTX-M producing
Enterobacteriaceae in farm animals and on meat products (Warren RE, 2008).
Why should antibiotic-resistant bacteria be showing up in animals? Please refer to a previous post.
We had
already shown these genes could occasionally be found in Salmonella spp. suggesting
blaCTX-M
genes were already
circulating in the community locally. However we had not actually demonstrated
their presence in food items yet (Koh TH, 2008).
We had
the opportunity to do this with 2 successive groups of Ngee Ann Polytechnic
students doing projects in our lab.
First,
Eugene and Si Xian looked at chicken meat since there already were many reports of ESBL-producing
Enterobacteriaceae in chickens in other countries. You can read a post on the blog
MIPHIDIC (click here) and the recently published
manuscript is open access (Lim EJ, 2016). To our surprise almost 80% of raw
chicken meat samples were positive for ESBL-producing Enterobacteriaceae.
Chicken meat from Malaysia had Entrobacteriaceae containing blaCTX-M-Groups 1,2, and 9, whereas
chicken meat from France and Brazil had blaCTX-M-Group
1, and blaCTX-M-Group 2
and 8 respectively. This is consistent with the published literature from
these countries.
Figure 2. Detection of ESBL-producing Enterobacteriaceae using ChromID ESBL plate. Red colonies suspected to be Escherichia. coli; Green colonies suspected to be Klebsiella spp.; Light brown colonies suspected to be Proteus spp.
Darris and Khai followed this up with a small study looking at beef and pork, also posted on MIPHIDIC (click here).
The yield in this case was
much lower. ESBL-producing Enterobacteriaceae were isolated from 4 pork
(originating from Australia and Indonesia) and 1 beef sample (originating from
Australia). Almost all isolates contained blaCTX-M
Group 1 except for an E. coli isolated from pork mince from
Australia that contained blaCTX-M
Group 8, This was interesting because whereas blaCTX-M Group 1 are the predominant ESBL in humans in Australia, blaCTX-M Group 8 is primarily
found in Brazil and I am not aware of it being described in Australia yet.
We
also isolated an MRSA from a pork sample but this was typed to ST3533-MRSA-IV,
spa type t015 which is a human rather pig-specific clone. This highlights a
limitation with this type of point-of-sale study-we cannot exclude human
contamination of the meat somewhere along the chain from farm to retail.
The
fact that we managed to find something, even with such a small sample size, was
in my opinion worth at least a short letter (perhaps in one of the countries
with a vested interest in our findings). Unfortunately the editors thought
otherwise. So rather than let the data remain hidden while we did a larger
study, we have put a manuscript up on a preprint (not peer-reviewed) server
where it is openly accessible to all (click here)
.
Figure 3. Actually,
I am inclined to make greater use of preprint servers. It gets your data out
into the scientific community quickly and you get some feedback on the usage of
your manuscript.
Going
back to ESBL-producing Enterobacteriaceae.
What is the situation in humans? In one hospital,
blaCTX-M Group 1 was predominant (Tan TY, 2010). This was representative of a nationwide 2008 survey of public sector hospitals in Singapore. Of 39 ESBL screen positive E. coli collected over 1 month, 67% (n=26) were
blaCTX-M Group 1, 18% (n=7) were
blaCTX-M Group 9. One isolate had both
blaCTX-M Group 1
and
blaCTX-M Group 9 and another had
blaCTX-M Group 2
and
blaCTX-M Group 9. Of 62 ESBL screen positive Klebsiella pneumoniae collected over the same period, 80.6% (n=50) were
blaCTX-M Group 1, three were
blaCTX-M Group 9
and
two were blaCTX-M Group 25 (unpublished data courtesy of Tan TY, Changi General Hospital and the NARSS group*). So the same
blaCTX-M Group
predominates in both humans and meat products locally.
This is not conclusive though because there are a number of different ESBLs represented in
blaCTX-M Group 1.
Bear in mind also,
even if you can find ESBLs in retail meat it does not necessarily follow that
spread to humans actually occurs.
In fact there is not that much literature providing
direct evidence of animal to human spread. This is possibly because the
sampling at both the retail meat or the human side is not extensive enough.
Also spread of resistance genes may occur by plasmid rather than bacterial
strain spread, and not many studies so far have been carried out at the plasmid
level. However there are a few studies that do show there is a link between
animals and humans at both the strain and plasmid level (Leverstein-van Hall
MA, 2011).
If animal meat to human
transmission is occurring, what is the mechanism as bacteria should not survive
the cooking process? It remains speculative, but cross-contamination may occur via
environmental surfaces and hands. It would be interesting to sample kitchen
sink drains, surfaces, and cleaning cloths in a variety of different settings across
Singapore (though a pilot on my sink at home was negative for ESBL-producing
Enterobacteriaceae).
*Network for Antimicrobial Resistance Surveillance, Singapore.
Tan
TY, Ng LS, He J, Hsu LY. CTX-M and ampC beta-lactamases contributing to increased
prevalence of ceftriaxone-resistant Escherichia coli in Changi General Hospital,
Singapore. Diagn Microbiol Infect Dis. 2010 Feb;66(2):210-3. (no free access)
In that post I alluded to the fact that my original
hypothesis was that people were getting GBS by eating raw tilapia in sushi and
sashimi as a result of a trade practice known as fish species substitution,
where a cheaper fish species is
substituted for a more expensive one and given a similar sounding name.
Somewhere on that plate is a
serotype Ia ST7 GBS.
The GBS outbreak eventually turned out to be due to
serotype III ST283 GBS found in bighead carp, giant snakehead and tilapia.
People were getting sick consuming raw fish dishes in local Chinese stalls
rather than Japanese-style restaurants.
However the question over whether GBS could be found
in sushi and sashimi continued to bother me. I got a chance to study this when
2 students from Temasek Polytechnic were attached to our laboratory in 2015 (Koh TH, 2017).
These students were required to do a small project so I set Terry and Fionicca off to
purchase sushi and sashimi from the cheaper Japanese-style food outlets and supermarkets
dotted across
Singapore. At the end of the day we collected 17 fish samples sold ready-to-eat. Twelve samples were salmon, 4
were sold as ‘Tai’, and one was actually sold as tilapia. The fish were
processed according the method detailed in a previous post.
We isolated 2 GBS. One was serotype II
ST1 GBS resistant to tetracycline from a Salmon sample. The other was serotype
Ia ST7 GBS susceptible to tetracycline from the tilapia sample. We were also able to find molecular evidence
for serotype Ia ST7 GBS in a sample sold as ‘Tai’ even though we were unable to
isolate the organism in culture. We did not find any serotype III ST283 GBS
though this may be a limitation of the small sample size.
Besides serotype III ST283 GBS, there are two other
types of GBS that are important fish pathogens, serotype Ib ST260 and serotype
Ia ST7.
Serotype Ib ST260 (originally named Streptococcus difficile) is the original
GBS pathogen of fish that was described as early as 1966. They are characteristically non-haemolytic. This type affects many species of fish and is
widely distributed. However, there is some data to suggest that this GBS type is not
found in Singapore, Malaysia, and Thailand, but may be found in Indonesia.
When I first started looking out for zoonotic GBS infections in 2008, I was
focused on serotype Ib ST260. In retrospect this was probably a mistake. This
type is so niche-adapted that it has a reduced genome content, and so far has
only been found in poikilothermic (cold blooded) animals like fish and frogs (Delannoy
CMJ, 2016). It has never been described in humans and probably poses no
zoonotic risk.
The origins of serotype Ia ST7 GBS are less certain.
This type has caused human infections in neonates in Japan
(Evans JJ, 2008), and adults in Iceland (Bjornsdotir ES, 2016), but on the
whole is rarely found in humans (note both Japan and Iceland have populations known
for high fish consumption). On the other hand, serotype Ia ST7 GBS have caused
many outbreaks in fish, especially tilapia, in asia and the middle east. This
type has also recently caused an outbreak locally in Singapore among farmed
golden pomfret (Trachinotus blochii) (Chong
SM, 2017). Interestingly in that manuscript, the authors say that GBS had not
been detected in cultured marine and freshwater fish in Singapore prior to
2014. To confuse matters, an old American type culture collection strain of
GBS (ATCC A909) was recently found to be serotype Ia ST7 (Tettelin H, 2005). This was isolated
from a human neonate with sepsis in 1934! The genome of ATCC A909 was recently
compared with that of serotype Ia ST7 GBS isolated from tilapia in China and
found to be similar (Liu G, 2013).
So is serotype Ia ST7 GBS a zoonosis or do fish get
infected as a result of human effluent polluting the environment? Delannoy et
al suggest that serotype Ia ST7 and serotype III ST283 GBS that cause fish
infections probably have a primarily homeothermic (warm blooded) host range and have
acquired fish-associated virulence factors not usually found in human or bovine
isolates (Delannoy CMJ, 2016). So perhaps an anthroponosis first then a zoonosis later?
What of the serotype II ST1 GBS resistant to
tetracycline from Salmon? We think this was probably the result of human
contamination of fish (human clones of GBS tend to be tetracycline resistant
whereas fish isolates tend to be tetracycline susceptible).
Going back to the issue of fish species substitution
and mis-labelling of fish. This is a widespread practice in the fish trade. Customers
may not know the subtle difference between dory fish (Vietnamese catfish Pangasius
spp. ) and the rather more prized John Dory (Zeus faber).
One of the more interesting parts of our study
was trying to identify the actual fish species that was being sold as ‘Tai’
which most people would probably assume was the fish you normally see on ‘Japan
Hour’* which is the Red Sea Bream (Pagrus major).
We did this by a method known as fish barcoding. This
involved sequencing the cytochrome c oxidase subunit I gene of the fish meat and
submitting the sequences to the Fish-Bol database.
Of the 4 ‘Tai’ samples, 2 were indeed Red
Sea Bream (Pagrus
major), 1 was Crimson Snapper (Lutjanus
erythropterus), this was the sample that had
molecular evidence for serotype Ia, ST7 GBS. The other sample was a Nile
tilapia (Oreochromis niloticus). So if you want to be sure of what fish you are eating, order the whole fish or go to a reputable restaurant (and be prepared to pay more).
*A popular TV programme in Singapore featuring travel and food in Japan.
Kalimuddin S, Chen SL, Lim CTK, Koh TH, Tan TY, Kam M, Wong CW, Mehershahi KS, Chau ML, Ng LC, Tang WY, Badaruddin H, Teo J, Apisarnthanarak A, Suwantarat N, Ip M, Holden MTG, Hsu LY, Barkham T; Singapore Group B Streptococcus Consortium. 2015 Epidemic of Severe Streptococcus agalactiae Sequence Type 283 Infections in Singapore Associated With the Consumption of Raw Freshwater Fish: A Detailed Analysis of Clinical, Epidemiological, and Bacterial Sequencing Data. Clin Infect Dis. 2017 May 15;64(suppl_2):S145-S152. (no free access)
Delannoy CM, Zadoks RN, Crumlish M, Rodgers D, Lainson FA, Ferguson HW, Turnbull J, Fontaine MC. Genomic comparison of virulent and non-virulent Streptococcus agalactiae in fish. J Fish Dis. 2016 Jan;39(1):13-29. (no free access)
Chong SM, Wong WK, Lee WY, Tan ZB, Tay YH, Teo XH, Chee LD, Fernandez CJ.Streptococcus agalactiae outbreaks in cultured golden pomfret Trachinotus blochii (Lacépède), in Singapore. J Fish Dis. 2017 Jul;40(7):971-974. (no free access)
