Multi-drug-resistant Gram-negative bacteria

The increasing prevalence of antibiotic-resistant Gram-negative bacteria is a serious concern not only for humans but also companion animals^1–3. Recent clinical attention has focused on the increasing frequency of Gram-negative pathogens responsible for hospital-acquired infections. In this group, extended-spectrum β-lactamase (ESBL) and carbapenemase producing Enterobacteriaceae, in particular E. coli and K. pneumoniae, as well as carbapenemase producing A. baumannii, have caused major challenges in the past decade. These three groups of organisms will be the focus of this article.

β-lactams are the most widely utilised antimicrobials worldwide for the treatment of infections in humans and animals. β-lactamase production is the most common mechanism of antibiotic resistance, particularly in Gram-negative bacilli. There are four different groups based on the Ambler molecular classification, which categorises the enzyme according to their amino acid sequence, primarily on their active site. Classes A, C and D are serine β-lactamases and Class B are metallo-β-lactamases which require a bivalent metal ion for activity, usually Zn²⁺ (Table 1)⁴.

Table 1. Classes of β-lactamase in Enterobacteriaceae and Acinetobacter spp.

Two types of cephalosporinases commonly described in human and animal pathogens are ESBLs and CMY-2 like. CMY-2 type of resistance was commonly described in Salmonella and E. coli isolated from food-producing animals^5,6. CMY-2 producing E. coli is also relatively common in humans and nearly as common as ESBL producing E. coli⁷. Nowadays, CTX-M type ESBLs are the most commonly reported β-lactamases both in human and animals. Until the year 2000, TEM and SHV type ESBLs were of great concern and mostly in E. cloacae and K. pneumoniae. However, since mid-2000s, there has been a dramatic shift into the predominance of CTX-M type ESBLs⁸. In dogs and cats, there have been reports of infections caused by CTX-M type ESBL producing E. coli associated with human infections^9,10. In the USA, CTX-M type ESBL, mostly CTX-M-15 occurred at the rate of 7.3% amongst E. coli causing urinary tract infections in dogs⁹ and caused serious infections, such as liver and bile infection in dogs¹⁰. CTX-M producing E. coli was quite common in pig farms in China, with the prevalence of 10.7%¹¹. The presence of this ESBL in farm animal causes a significant threat to public health.

E. coli is a leading cause of urinary tract infections in humans and dogs. E. coli can be divided into four phylogenetic groups: A, B1, B2 and D. The virulent extra-intestinal pathogens are usually found in classes B2 and D¹². In the last decade, E. coli sequence type (ST) 131 has emerged as a pandemic uropathogenic E. coli causing community and hospital-acquired infections especially urinary tract infections^13,14. This clone potentially can harbour a variety of β-lactamase genes; however, it is most strongly associated with CTX-M-15 ESBL. In addition, the pandemic clone E. coli ST131 also belonged to the phylogenetic group B2, the highly virulent group of ExPEC. The combination of virulence and antimicrobial resistance may give E. coli ST131 a fitness advantage over other E. coli strains, which highlights the widespread and successful dissemination of E. coli ST131.

E. coli ST131 is also represented among resistant isolates in companion and farm animals. A European study determined the presence of E. coli ST131 which mostly produced CTX-M-15, comprising 5.6% of ESBL-producing E. coli isolates recovered from companion animals¹⁵. In Australia, E. coli ST131 accounted for 7.2% of isolates from companion animals¹⁶. Transmission between dogs and cats sharing the same clone, E. coli ST131, has been described previously¹⁷. Dogs in particular have been identified as a possible reservoir not only for ST131, but also for extra-intestinal pathogenic E. coli (ExPEC) in general for the transmission to other pets and humans¹⁸. E. coli ST131 has also been reported from poultry and pig farms in Spain¹⁹. In this instance, both companion and farm animals can be the reservoirs for E. coli ST131 and other cephalosporin-resistant E. coli. Studies on retail meat for human consumption have found E. coli indistinguishable from human E. coli causing urinary tract infections (UTIs) and E. coli ST131 was isolated from chicken²⁰. Further studies are urgently needed to investigate the pathways of transmission between humans and animals to determine whether isolates found in retail meat and livestock pose an imminent threat to humans.

Various Enterobacteriaceae confer antibiotic resistance by producing enzymes such as carbapenemases. The carbapenemase modifies the carbapenems through the hydrolysis of the antibiotic and has been defined into different classes using the Ambler classification system. The resistance in Enterobacteriaceae is found within Ambler class A (KPC – K. pneumoniae Carbapenemase), class B (metallo-β-lactamases) and class D (OXA – oxacillinases) (Table 1)²¹. KPC-type β-lactamases, are the most frequent cause of carbapenem resistance in the United States and has spread rapidly to Europe^22,23. The most recent class B enzyme identified is the New Delhi Metallo–β-lactamase (NDM-1) in 2009²⁴. These enzymes are frequently found in K. pneumoniae and E. coli. The majority of NDM-producing isolates are found on the Indian subcontinent²⁵.

The recent emergence of CRE represents a significant threat and has also drawn substantial media attention, especially through the spread of the NDM-1²⁶. The NDM-1 gene was first described from a Swedish patient who travelled to India in 2008, but this gene has now spread worldwide²⁵. NDM-1 can be found in numerous species of Enterobacteriaceae, including E. coli and various species of Acinetobacter spp.²⁷. NDM gene is able to spread rapidly by residing in self-transferrable plasmids. NDM-1 producers have been isolated from water and other environmental samples in India²⁸. A total of 88 NDM-1 cases were reported in the United Kingdom (up to March 2011²⁹); 13 NDM-1 cases were reported in the USA³⁰. Eight cases have been reported in Australia. Most cases identified outside India and Pakistan, including those in Australia and New Zealand, were related to travel and/or medical treatment in India or Pakistan^24,31. There have been several cases due to local acquisition of the NDM producers or without travel history in Europe and Asia^32,33. The rapid spread of NDM has moved outside the hospital. Studies in China found NDM-1 producing Acinetobacter spp. from hospital waste³⁴ and food of animal origin³⁵. The worldwide spread of NDM producers in hospitals and the environment are clearly a real threat.

A. baumannii has gained recognition as a major nosocomial pathogen in recent years³⁶ predominantly affecting immunocompromised or critically ill patients. It causes a wide range of infections including pneumonia, bacteraemia and infections of the skin, bone, urinary tract and central nervous system³⁶. The remarkable ability of A. baumannii to up-regulate or acquire antibiotic resistance determinants makes the bacterium a significant nosocomial pathogen. Resistance to carbapenems has been reported worldwide, including Australia^37–39. Many genotypes conferring such resistance have been found to belong to European clones (EU) I, II or III³⁷. Although several molecular mechanisms were responsible for conferring carbapenem resistance, the most common is the production of class D β-lactamases, OXA-type carbapenemases. Class B metallo-β-lactamases (such as IMP-, VIM- and SIM-types) are occasionally identified in A. baumannii isolates which have acquired these resistant determinants via class 1 integrons (Table 1)⁴⁰. Alteration of penicillin-binding proteins and loss of outer membrane proteins⁴¹, efflux pump mechanisms and other β-lactamases are also found in A. baumannii⁴².

The OXA-type carbapenemases identified in A. baumannii include both (i) acquired types (OXA-23-, OXA-24- and OXA-58-like) where their gene clusters have been found either in the chromosome or plasmid and (ii) naturally occurring chromosomal OXA-51-like⁴³. Multiple studies regarding genomic sequences surrounding these genes revealed the pivotal role of insertion sequence (IS) elements. The presence of ISAba1 upstream of bla_OXA-23 as well as intrinsic chromosomal bla_OXA-51-like provide promoter sequences consequently leading to the over-expression of these downstream genes^40,44. ISAba1 and other IS elements, such as ISAba2, ISAba3 and IS8 were shown to enhance the expression of bla_OXA-58⁴⁵. These IS elements play significant roles in the expression of various OXA-genes in A. baumannii. A. baumannii can also be isolated from faeces of slaughtered pigs and cattle⁴⁶. These strains from animals possessed bla_OXA-51-like, however, the resistance islands could not be found and none of the isolates from animals belonged to the European clones I, II and III as the common clones of human A. baumannii strains⁴⁶.

The rapid spread of multi-drug-resistant bacteria Gram-negative bacteria, especially in nosocomial pathogens; in the past decade underline the importance of infection control precautions in both human and veterinary hospitals. The possibility that farm animals may be the reservoir of resistance mechanisms and the current evidence of spread of the most recent and notorious carbapenem resistance genes demonstrate the need for increased surveillance of antibiotic usage in veterinary and farm animals as well as in humans.